Genetics, Genomics & Mutations

DNA & GENES

DNA (deoxyribonucleic acid) serves as the blueprint for who we are, what we're made up of, and how our body functions on a (cellular) level. We inherit DNA from our biological parents, half from one parent and half from the other. DNA can be found in the nucleus of every human cell. 

In humans, sequences of DNA, called genes, are transcribed into RNA which then codes for the production of specific proteins (protein synthesis) that influence various biological processes within the body. These proteins are each responsible for specific functions in the body to keep things running smoothly, but when the gene they arise from is mutated, the activities the proteins are responsible for are impacted as well. 

DNA--> RNA--> protein --> impacts a biological function/process

The human genome contains roughly 100,000 unique genes and, of those, about 6000 genes have been determined to have an effect on or be associated with disease risk or development. (source)

Related Terminology:

• Gene/Genetic: a gene is a sequence of DNA, found at a specific location on a chromosome, that codes for the production of a protein. 

• Coding/Protein-Coding genes: According to the National Human Genome Research Institute, humans have about 20,000 genes that code for proteins.

• Non-coding genes: these genes do not code for the production of proteins (that impact how the body functions on a cellular level); the large majority of the human genome is made up of non-coding DNA

• Genome/Genomic: the genome is the complete collection of genes in an organism and genomics is the study of the functions of those genes. 

• Chromosomes: composed of DNA, containing all of a person's genes. Each person typically has 46 chromosomes (23 pairs of chromosomes from each parent.) Genes are located at specific spots (loci) on specific chromosomes. DNA is the nucleic acid of chromosomes. 

• Amino acid: building block of proteins, make up proteins

• Protein: molecule that directs and influences biological processes in the body

• Proteomics: the study of proteins and how they function in the body

• Nucleic acid: building block of DNA and RNA; compounds made up of a chain of nucleotides that contain information that directs protein synthesis and cellular processes. 

• Nucleotide: is made up of a sugar (deoxyribose in DNA), a phosphate, and a nitrogen base. There are four possible DNA nitrogen bases: adenine (A), thymine (T), guanine (G), and cytosine (C). In RNA, the thymine (T) is replaced with uracil (U). Nucleotides bond to form nucleic acids. Adenine and thymine pair with each other using two hydrogen bonds, and cytosine and guanine pair with each other using three hydrogen bonds to create nucleic acids that form the double-helix structure of DNA. 

Sugar + phosphate + nitrogen base = nucleotide

ribose (sugar) + phosphate + A, U, C,  or G = RNA nucleotide

deoxyribose (sugar) + phosphate + A, T, C, or G = DNA nucleotide

Chain of nucleotides = nucleic acid= DNA or RNA

MUTATE TO SURVIVE

When cells replicate and divide, the genetic code of that cell has to be duplicated to create new “daughter” cells. Often, mistakes are made during this process and these mistakes, called mutations, can lead to changes in the cell’s genetic makeup and how the cell functions in the body. It's estimated that trillions of mutations occur in our bodies daily, but we have built-in mechanisms to automatically repair these mistakes. However, if a mutation occurs in a gene that is responsible for repairs (like DNA repair genes), the gene won’t be able to correct a mutation on another gene. Driver mutations, or genetic changes that initiate and proliferate cancer, often occur in DNA repair genes or tumor suppressor genes (TSG), genes responsible for preventing the development & escalation of cancer. When these genes are mutated and don’t function as they should, cells can grow uncontrollably, leading to cancer. 

The mutations that lead to cancer initiation and cancer proliferation (driver mutations) are typically those that interfere with cell signaling as it relates to cell division and growth. These mutations mess up, block, or amplify signals, and tell our cells to do things that they typically wouldn't do, including to continue dividing and replicating unchecked. These genes, when not mutated, would carry out activities that allow the cell to reach or maintain homeostasis--a state of balance. But when they are mutated, the system of checks-and-balances no longer functions properly. 

At the cellular level, mutations can affect:

• development of cancer
• rate of growth/proliferation of cancer
• angiogenesis
• invasion/metastasis
• immune system (suppression, evasion)
• therapy resistance

There are two types of mutations you may encounter:

1. Hereditary mutations, also called germline or inherited mutations, are genetic changes you are born with that were passed down from your parents. Some hereditary mutations make cancer development more likely, and if you have one of these mutations, it may also affect your parents, siblings, or children and their risk of developing cancer. A genetic test, or hereditary cancer panel, is used to identify these familial mutations.  If you haven’t had a genetic test to identify hereditary mutations, ask your doctor for more information. Most insurance plans will cover this testing based on your type of cancer, stage, and/or family history. If you’re not covered, visit the Cost of Care page on our website for a list of organizations offering financial aid for genetic testing. It’s important to consider this type of testing because some mutations may make you eligible or ineligible for treatments beyond the traditional chemo, surgery, and radiation offerings.  Additionally, if you test positive for an inherited mutation, your family may need to be notified and/or tested as well. This testing (testing patient's family after patient receives a positive genetic test for a hereditary mutation) is called cascade testing. Here's a <2-minute video that gives an overview of cascade genetic testing, explained by a Genetic Counselor. 
2. Acquired mutations, also called somatic or sporadic mutations, are genetic changes that occur over the course of your life and are not present in all cells in your body. The acquired mutations being sought by your provider are those specific to the genes in your tumor, not your entire body. You may hear tests for these mutations referred to as genomic profiling, genome sequencing, tumor sequencing, tumor subtyping, molecular profiling, or biomarker testing. Cancer is known to mutate regularly (think about how the flu has new variants all the time) so it's worth exploring with your doctor if your tumor should be profiled again during your course of care in case new mutations arise that may be targetable by other therapies. 

Mutations in these three types of genes are most often behind the development of cancer: 

Tumor suppressor genes (TSG):  These genes help regulate growth by slowing down cellular division and promoting apoptosis (programmed cell death). Essentially, they work to prohibit the development of tumors (cluster of overgrown cells). When TSGs are mutated, cells can grow out of control and become cancerous.

• TP53, PTEN, APC, BRCA1, BRCA2, CHEK2, RB1
• "TSG [tumor suppressor gene] p53 is known as the guardian of the genome. It activates proteins that puts brakes on cell division. It is our most prominent intracellular defender against cancer...Indeed, p53 is the most commonly mutated gene in many types of cancers." -Azra Raza, MD, The First Cell

DNA repair genes: Do exactly what you would expect--they make repairs to DNA. If repairs are not sufficient to resolve the issue, DNA repair genes can trigger apoptosis to kill off irreparable cells. When DNA repair genes are mutated, repairs cannot be made as usual and this can lead to more mutations and the development of cancer. 

• BRCA1, BRCA2, TP53, RB1, MMR (mismatch repair)

Oncogenes: Proto-oncogenes are useful genes we have in our bodies that control cellular growth and replication. However, if one of these genes becomes mutated, it won't work properly and can encourage continued growth and proliferation, leading to tumor development. Once proto-oncogenes are mutated or activated, they are called oncogenes. 

• Ras family (KRAS, NRAS, etc.), HER2/ERBB2, Myc (CMYC, NMYC), EGFR, BCR-ABL, PIK3CA, ALK

Related Terminology:

• Driver mutations- the changes in the tumor DNA that causes something (that -when unmutated-controls cancer growth/proliferation in our body's cells) turn on or off and led to cancer developing 

• Targetable mutation: a mutation for which there is a drug (called a targeted therapy) designed to offset that mutation; these therapies often have high response rates for patients with those mutations and poor/no response for patients lacking the mutation. Can also be called a medically actionable variant/mutation. 

• Wildtype: this refers to a gene that is NOT mutated in your cancer; for example if your KRAS is defined as wild-type (WT), then your KRAS gene is not mutated.

• Variant: a type of change in a gene, a type of mutation; different types of mutations can occur within a single a gene. Certain variants are linked to cancer/disease (pathogenic variant) and some variants are not yet determined (VUS- variant of uncertain significance) 

• SNP (single nucleotide polymorphisms): a change in a single nucleotide (A, T, G, or C) 

• Tumor Mutational Burden (TMB) is a biomarker used to measure the estimated quantity of mutations found within a tumor's DNA. This value (mutations per megabase of DNA or mut/Mb) can be determined most accurately by analyzing biopsied tumor tissue using next-generation sequencing (NGS) or whole-exome sequencing (WES), though less-invasive blood tests to assess TMB are currently being researched. TMB is usually reported as TMB-high, TMB-intermediate, or TMB-low, but some reports may provide specific values like 5 mut/Mb or 21 mut/Mb. Whether a specific value falls into the low, intermediate, or high classification is dependent upon the cancer type. TMB is most often used to identify tumors that may be susceptible (or respond well) to immunotherapy, and can inform prognosis for some cancer types. TMB-high is most often indicative of a tumor that may respond to immunotherapy.  

• Next-Generation Sequencing (NGS or Next-Gen Sequencing)- a panel that tests for multiple blood-based or saliva-based biomarkers (mutations, tumor markers, proteins, etc.) at the same time. 

How does DNA drive activity in the body? PROTEINS!

In humans, sequences of DNA, called genes, are transcribed into RNA which then codes for the production of specific proteins (protein synthesis). These proteins are each responsible for a specific function(s) in the body to keep things running smoothly, but when the gene they arise from is mutated, the activities the proteins are responsible for are impacted as well. 

DNA--> RNA--> protein --> impacts a biological function/process

When a gene is mutated, its proteins can be impacted in different ways.  

• The protein may not be produced at all which means the activities for which it was responsible do not occur, which can lead to problems. 
• Too little of the protein may be produced which means the activities don't occur as often, as quickly, or as thoroughly as they should, which can lead to problems. 
• Too much of the protein may be produced which means the activity takes place more often, more quickly, or more heavily than it should, which can lead to problems. 

If a protein, for example, is responsible for slowing down or stopping (cellular) reproduction, and it is not produced or too little of it is produced, then cells may replicate faster than they should, which can lead to cancer. Similarly, if a protein is responsible for keeping cells stuck together and it is not produced, then cells can start to break away from each other and potentially travel to other areas of the body. If the cells in question are tumor cells and they become "unstuck," it can potentially lead to the circulation of tumor cells and, eventual, metastasis. 

Take a look below to learn more about what some proteins are responsible for in the body and with which gene mutations they're associated. 

Proteins of some commonly-mutated genes in cancer and what they do in the body: 

• BRCA1 proteins: "has multiple functions in different cellular processes, including DNA repair, transcriptional activation, cell cycle regulation and chromatin remodeling."
• BRCA2 proteins: "plays a role in transcriptional and cell cycle regulation, DNA repair, mitophagy and stabilization of replication fork."
• CHEK2 protein: "regulates cell division, can stabilize tsg p53 protein, allows BRCA1 to restore survival after DNA damage"
• PTEN proteins: "functions as a tumor suppressor by negatively regulating AKT/PKB signaling pathway"
• TP53 proteins: "responds to diverse cellular stresses to regulate expression of target genes, thereby inducing cell cycle arrest, apoptosis, senescence, DNA repair, or changes in metabolism."
• APC protein: "that acts as an antagonist of the Wnt signaling pathway. It is also involved in other processes including cell migration and adhesion, transcriptional activation, and apoptosis."
• BRAF protein: "plays a role in regulating the MAP kinase/ERK signaling pathway, which affects cell division, differentiation, and secretion."
• HER2 protein "involved in negative regulation of transcription."
• PIK3CA protein: "Participates in cellular signaling in response to various growth factors. Involved in the activation of AKT1 upon stimulation by receptor tyrosine kinases ligands such as EGF, insulin, IGF1, VEGFA and PDGF. Involved in signaling via insulin-receptor substrate (IRS) proteins. Essential in endothelial cell migration during vascular development through VEGFA signaling, possibly by regulating RhoA activity. Required for lymphatic vasculature development, possibly by binding to RAS and by activation by EGF and FGF2, but not by PDGF. Regulates invadopodia formation through the PDPK1-AKT1 pathway. Participates in cardiomyogenesis in embryonic stem cells through a AKT1 pathway. Participates in vasculogenesis in embryonic stem cells through PDK1 and protein kinase C pathway."
• PIK3CA protein: "The PIK3CA gene holds the instructions for making a protein called p110a. This protein is important for many cell functions, including telling your cells when to grow and divide. Certain people may have mutations in this gene. PIK3CA gene mutations cause cells to grow uncontrollably, which can lead to cancer."
• PALB2 protein: "This protein binds to and colocalizes with the breast cancer 2 early onset protein (BRCA2) in nuclear foci and likely permits the stable intranuclear localization and accumulation of BRCA2" 
• MLH1 protein: "The encoded protein is also involved in DNA damage signaling" 
• MSH6 protein: "The encoded protein heterodimerizes with MSH2 to form a mismatch recognition complex that functions as a bidirectional molecular switch that exchanges ADP and ATP as DNA mismatches are bound and dissociated."
• EPCAM (epithelial cell adhesion molecule) protein: "This gene encodes a carcinoma-associated antigen and is a member of a family that includes at least two type I membrane proteins."

Within each gene, there are many possible point mutations that can occur. Each point mutation can have a different effect: positive, negative, neutral, or unknown. 

Types of Point Mutations:

• nonsense (change in one base pair; sends message to stop building protein early so the protein may not work properly or at all) 
• deletion (piece of DNA is taken out which could affect how protein functions)
• insertion (piece of DNA is added in which could affect how protein functions)
• missense (one amino acid is substituted for another in one base pair)
• silent (don't affect amino acid sequence so they don't affect resulting protein)
• frameshift (changes the reading frame of gene, shift, resulting protein is usually nonfunctional; insertions, deletions, and duplications can all be frameshift mutations) 
• duplication (piece of DNA is copied and added, effects resulting protein's function) 

Genomic Instability

At some point between your initial diagnosis and beginning treatment, your tumor(s) may be tested for genomic instability. This can be done a number of ways depending on the type of cancer and what specific abnormalities are being measured within the cancer's genome. Ask your doctor if your cancer needs to be tested for genomic instability, how it will be tested, and what the results could imply for your diagnosis or treatment. 

The National Institutes of Health (NIH) defines genomic instability as "The increased tendency for DNA mutations (changes) and other genetic changes to occur during cell division. Genomic instability is caused by defects in certain processes that control the way cells divide. It occurs in many types of cancer. These defects may include mutations in certain genes involved in repairing damaged DNA or mistakes that don’t get corrected when DNA is copied in a cell. They may also include defects such as broken, missing, rearranged, or extra chromosomes. Studying genomic instability may help researchers understand how certain diseases, such as cancer, form. This may lead to new ways to diagnose, treat, and prevent disease."

Genomic instability, simply speaking, means the genetic makeup of the cancer (its genome) is wonky, unstable, and carries a bunch of mutations. As these mutations build up, they invite more mutations and replication errors, and the cancer's genome becomes increasingly unstable. As instability escalates, often due to mutations in genes that keep the genome in check, cancer is emboldened and better able to do what it wants to do—proliferate and spread. Hanahan and Weinberg identified "genomic instability" as an emerging Hallmark of Cancer in their 2011 paper, pointing out that, "instability of the genome is inherent to the great majority of human cancer cells," Given that genomic instability is so prevalent across cancer types and can lead to cancer progression and poorer patient prognoses, it's an important feature of cancer to keep an eye on. Additionally, and more optimistically, a status of genomic instability in some cancers can open the door for treatment with immunotherapy. It's theorized that the more unbalanced a cancer genome is, the easier it is for the immune system to identify, and subsequently attack, those cancer cells. 

"Genomic instability is...a driving force of tumorigenesis in that continuous modification of tumor cell genomes promotes the acquisition of further DNA alterations, clonal evolution, and tumor heterogeneity. It is a feature of almost all cancers and has been observed in a range of malignant stages, from pre-neoplastic lesions prior to acquired TP53 mutations to advanced cases," per a 2013 article in Cancer and Metastasis Reviews by Pikor, L. et al. The authors went on to explain, "Genomic instability refers to a variety of DNA alterations, encompassing single nucleotide to whole chromosome changes, and is typically subdivided into three categories based on the level of genetic disruption. 

• Nucleotide instability (NIN) is characterized by an increased frequency of base substitutions, deletions, and insertions of one or a few nucleotides
• Microsatellite instability (MIN or MSI) is the result of defects in mismatch repair genes which leads to the expansion and contraction of short nucleotide repeats called microsatellites
• Chromosomal instability (CIN) is the most prevalent form of genomic instability and leads to changes in both chromosome number and structure. 

While instability is a characteristic of almost all human cancers, cancer genomes vary considerably in both the amount and type of genomic instability they harbor. Importantly, the instability phenotype has implications in patient prognosis as well as patient management, specifically with the choice of therapeutic agents."

According to a 2017 review in Cancer Research, "Genomic instability has previously been associated with poor prognosis. However, we have evidence that for solid tumors of epithelial origin, extreme levels of genomic instability, where more than 75% of the genome is subject to somatic copy number alterations, are associated with a potentially better prognosis compared with intermediate levels under this threshold.  Another possibility is that tumors with high levels of genomic instability are more immunogenic than other cancers with a less extensive burden of genetic aberrations." Later in the review, the authors mentioned, "Measuring a tumor's degree of genomic instability prior to therapy may be informative for assessing chemotherapy."