Mutational Analysis

Mutational Analysis

• Testing for the presence of a specific mutation or set of mutations, as opposed to complete gene sequencing or mutation scanning, which detect most, if not all, mutations in the tested region.

Types of Mutations

Mutations that can occur in all genetic disorders

• Missense mutations- arise from single base pair [bp] substitutions that cause a change in the amino acid sequence.

• Nonsense mutations- arise from single base pair substitutions; which cause the creation of a premature stop codon.

• Splice mutations- alters one of these signals, then part of or the whole intron may not be removed and remains as part of the final RNA molecule.

• Frameshift mutations- one or more bases are inserted or deleted. (deletions or insertions can be ‘in-frame’ or ‘out-of-frame’.)

• Wild-type sequence [part of the human antithrombin gene]

Glu Leu Ser Lys Ala Asn 5’- GAA CTG TCC AAG GCC AAT

• Mutant Sequence: in-frame 3bp [CTG] deletion

Glu Ser Lys Ala Asn 5’- GAA TCC AAG GCC AAT

• Wild type sequence

Glu Leu Ser Lys Ala Asn 5’- GAA CTG TCC AAG GCC AAT

• In this sequence if we delete the CT in the CTG that encodes leucine then this changes the reading frame and the corresponding amino amino acid sequence

Glu Val Gln Gly Gln ... 5’- GAA GTC CAA GGC CAA T..

• Major gene deletions/insertions- loss of one or more genes depending upon the size of the event

Single Nucleotide Polymorphisms (SNPs)

• most common type of genetic variation among people

• represents a difference in a single DNA building block, called a nucleotide

• occur normally throughout a person’s DNA• act as biological marker• occur within a gene or in a regulatory region

near a gene

• may play a more direct role in disease by affecting the gene’s function

• help predict an individual’s response to certain drugs, susceptibility to environmental factors such as toxins and risk of developing particular diseases

Allele Specific Oligonucleotide (ASO)

• short piece of synthetic DNA complementary to the sequence of a variable target DNA.

• typically an oligonucleotide of 15–21 nucleotide bases in length.

• designed (and used) in a way that makes it specific for only one version, or allele, of the DNA being tested.

• designed to detect a difference of as little as 1 base in the target's genetic sequence, a basic ability in the assay of single-nucleotide polymorphisms (SNPs)

Allele-Specific Real Time-Polymerase Chain Reaction

• application in which alleles that differ by one or more nucleotides can be distinguished on the basis of PCR amplification

• detection of any mutation in human DNA• used to identify single nucleotide

polymorphisms (SNPs)• uses primers whose 3’ end encompasses the

SNP

Epidermal Growth Factor Receptor (EGFR)

• also known as ErbB 1 and HER1• is a cell-surface receptor that binds epidermal

growth factors, resulting in the activation of various signaling pathways inside the cell

• EGFR signaling pathway causes cell growth and proliferation via several signaling molecules, including KRAS and BRAF.

EGFR

v-raf murine sarcoma viral oncogene homolog B (BRAF)

• located on the long (q) arm of chromosome 7 at position 34

• composed of 18 exons, and the most common activating mutation is found in exon 15 at nucleotide position 1799

• provides instructions for making a protein that helps transmit chemical signals from outside the cell to the cell's nucleus

• part of a signaling pathway known as the RAS/MAPK pathway, which helps control several important cell functions

• belongs to a class of genes known as oncogenes

• BRAF gene mutation testing has emerged as an important tool for diagnosis, prognosis, treatment, and predicting patient outcome in response to targeted therapy.

• In treatment decisions, because in patients with advanced colorectal cancer, BRAF mutation testing has been suggested since its presence confers resistance to epidermal growth factor inhibitors (cetuximab and panitumumab), when k-ras is not mutated.

RAS/MAPK pathway

• regulates the growth and division (proliferation) of cells, the process by which cells mature to carry out specific functions (differentiation), cell movement (migration), and the self-destruction of cells (apoptosis)

• chemical signaling through this pathway is essential for normal development before birth

Kirsten rat sarcoma viral oncogene homolog (KRAS)

• located on the short (p) arm of chromosome 12 at position 12.1

• provides instructions for making a protein called K-Ras that is involved primarily in regulating cell division

• also part of the signaling pathway RAS/MAPK pathway, which the protein relays signals from outside the cell to the cell's nucleus

• these signals instruct the cell to grow and divide or to mature and take on specialized functions (differentiate).

• oncogene• KRAS gene is in the Ras family of oncogenes,

which also includes two other genes: HRAS and NRAS

• proteins produced from these three genes are GTPases

Interpretation