Wk6 Mechanisms And Effects Of Mutations

Question 1

What are variations in the gene?

Answer 1

• Alterations in the sequence of bases in a specific section of DNA
– Single nucleotide polymorphisms
– Small deletions or duplications (few bases)
• Microsatellites (tandem repeats of 2-6 bp) - <100 bp in
• Minisatellites (“variable number
tandem repeats” of 10-60 bp) – can span several kb
• Larger deletions/duplications (copy number variation) of DNA segment - may include one to many genes (1kb – 1 Mb)
• Changes in the number or structure of chromosomes

Question 2

What are altered effects of a protein or control of genes?

Answer 2

Normal human variation
- eye colour

Differences in response to medication
- effects of antidepressants

Influence likelihood of disease
- diabetes

Directly result in a genetic condition
- sickle cell disease

Question 3

How are genome variants?

Answer 3

Chromosomal rearrangements LARGE - SMALL single base changes

single nucleotide polymorphisms in population COMMON - RARE mutation in a gene in one family

Non-harmful polymorphisms NON-PATHOGENIC - PATHOGENIC disrupt gene function

Question 4

What is a mutation?

Answer 4

an alteration or change in the genetic material
• In clinical use usually = “harmful”
• from exposure to mutagenic agents but more arise spontaneously through errors in DNA replication/repair
• more likely to be recognised if effects are detrimental

Question 5

What are DNA sequence variants?

Answer 5

• Mutation – harmful sequence variant alters gene function and phenotype

• Polymorphisms – non harmful:
sequence variant is in non-functional DNA

sequence variant is within gene but does not change amino acid

sequence variant changes amino acid but does not alter protein function

SNP – single nucleotide polymorphism (commonest type of variant)

Question 6

What are single nucleotide polymorphisms?

Answer 6

Can occur throughout the gene
Most common type of genome variation
To be called an SNP a base change has to have a fréquence of >1%

Question 7

How can the genome be examined?

Answer 7

Bases - sequencing and microarray analysis

Large blocks of DNA - microarray analysis and fluorescence in situ hybridisation (FISH)

Chromosomal - light microscopy

Question 8

How is DNA sequenced?

Answer 8

• The dideoxy, or chain termination method
• Developed by Fred Sanger in 1977 (won him a Nobel Prize)
 Amplify very small amounts of target DNA (usually by PCR)
 DNA is used as a template to generate a set of fragments that
differ in length from each other by a single base.
 The fragments are then separated by size, and the bases at the end are identified, recreating the original sequence of the DNA.

Question 9

Why sequence DNA?

Answer 9

sequencing determines the exact position of the mutation
within the gene
determines the type of the mutation (including single base changes)

Question 10

How is nucleic acid sequencing by chain termination is not automated?

Answer 10

the use of fluorescently-labelled ddNTP terminators has allowed automation and high-throughput sequencing
– this technical advance was essential for the rapid completion of the Human Genome Project
a different fluorescent labelling molecule (“fluorophore”) is used for each of the ddNTPs, each with a different emission colour
all four sequencing reactions can be carried out simultaneously in a single tube,

Question 11

When do mutations occur?

Answer 11

1. Cell division

2. From intrinsic and extrinsic attacks on DNA

Question 12

What causes mutations - endogenous mechanisms causing DNA damage?

Answer 12

• Endogenous mechanisms causing DNA damage
– Depurination (spontaneous fission of link between purine base and sugar. Causes loss of adenine or guanine from helix – deletion of base or incorrect nucleotide in new strand)
– Deamination (cytosine deaminates to uracil – causing substitution of an A in new strand)
– Reactive oxygen (attack purine/pyrimidine rings)
– Methylation of Cytosines at CpG dinucleotides spontaneous deamination of 5-methyl-cytosine to thymine

Question 13

What is a common mechanism of mutation?

Answer 13

C —> T at CpG

• high frequency of C —> T transitions in the genome
• especially at CpG dinucleotides
– CpG to TpG mutations = 30-40% of all point mutations
• cytosines at CpG sequences are frequently methylated; 5-methyl-cytosine can spontaneously deaminate to thymine
• mutation rate at CpG is 8.5 times that of other dinucleotides
• frequent effect is production of a nonsense mutation CGA —> TGA
arginine —> stop codon

Question 14

What causes mutations - extracellular agents causing DNA damaged?

Answer 14

– Ultraviolet light (cross-linking of adjacent thymines on
a DNA strand to form a stable dimer)
– Environmental chemicals (interpolate into DNA or cause DNA breaks or chromosome aneuploidy)
– Ionizing radiation (causes breaks in DNA)

Question 15

What causes mutations - thymine dimmers as a result of UV radiation in sunlight?

Answer 15

Two adjacent thymine bases covalently attach to each other.

Thymine dimers disrupt 3D structure, and can stall the DNA replication machinery

Bases damaged by sunlight (and by chemicals) are excised and the strand re-synthesised

Question 16

What is a germ line mutation?

Answer 16

• present in egg or sperm

- heritable

Question 17

What is a somatic mutation?

Answer 17

• occurs in non-germ line tissues

- nonheritable

Question 18

How to correct DNA replication errors?

Answer 18

DNAreplication machinery has proof reading:
DNA polymerase adds a base, checks it, excises it if it is wrong and then moves on.

• DNA mismatch repair is backup system which corrects 99% of residual errors from replication machinery
• Replication copy errors leave mispaired nucleotides – would cause a permanent mutation when the strand with the error is copied in the next round
• Protein complex recognises DNA mismatch, excises newly synthesised mismatched strand and uses original template strand to re-synthesise new strand

• Mutations in the mismatch repair genes themselves lead to accumulation of somatic mutations and so predispose to cancers

Question 19

How to correct endrogenous damage to DNA?

Answer 19

• Mismatch identified

* Usually repaired by excision repair mechanisms (130 genes involved!)

Question 20

How to correct damage to DNA - due to ionising radiation and reactive oxygen species?

Answer 20

DNA double strand breaks repaired either by
• The accurate method of using a sister DNA molecule (homologous recombination)

• Or by end-joining broken ends (error prone)

Question 21

What are the types of mutations?

Answer 21

Usual code
Missense
Nonsense
Frameshift
Duplication
Deletion
Insertion

•Varying effects on health depending on where they occur and whether they alter the function of essential proteins

Question 22

What are pathological mutations associated with protein coding genes?

Answer 22

• DNA coding sequence of a gene
• intragenic non-coding sequences
necessary for correct gene expression
• regulatory sequences

Question 23

What is a silent mutation/sequence variant?

Answer 23

A base pair change that does not change the amino acid sequence

– No alteration in polypeptide composition of protein
– Single base substitution alters genetic code but not amino acid
– Single base substitution at third position of a codon may have no effect eg GGA → GGC or GGG or GGU: all glycine
– Most frequent type in coding DNA as effect neutral and not subject to selection (basis of single nucleotide polymorphisms – SNPs)
– Can create a cryptic splice site

Question 24

What is a missense mutation?

Answer 24

Changes to a codon for another amino acid (can be a harmful mutation or a polymorphism with neutral effect)

– Single base substitution codes for a different amino acid
– If chemically dissimilar, structure and function of protein altered
– If chemically similar, biological activity may not be affected (conservative substitution)

Question 25

Nonsense mutations

Answer 25

Change from an amino acid codon to a stop codon

– Creates a termination codon: UAA, UAG, UGA
– Allele with mutation unlikely to retain normal activity:
• Stop codon results in premature termination of translation → truncated protein
• Aberrant transcript usually degraded by “nonsense mediated decay” process

Question 26

What is a splice-site mutation?

Answer 26

A change that results in altered RNA sequence

Question 27

What is a summary of splice-site mutations?

Answer 27

• changes in introns, and intergenic regions may not affect the phenotype
• but if splice-sites become mutated:
– substantial changes in both the size and the content of a protein can be caused by splice site mutations; common
• normal splicing of RNA depends upon the presence of the GU and AG donor and acceptor sites at the 5’ and 3’ ends of the intron
• mutations of the splice site will prevent the correct splicing of pre-messenger RNA, subsequently producing an altered protein

Question 28

Frameshift mutations

Answer 28

Insertion or deletion of base pairs, producing a stop codon downstream

– Insertion or deletion of nucleotides not a multiple of three
– Amino acid sequence following mutation different from intended sequence
– Usually leads downstream to a new stop codon
– A shortened altered protein may be expressed or mRNA degraded by nonsense mediated decay system

Question 29

What are copy number variants?

Answer 29

• Small arrays of triplet repeats (eg CAG) in coding sequences of genes are prone to expand in number and disrupt the function of the gene
• Short tandem repeats can mispair and cause pathogenic deletions and insertions which cause a frameshift
• Expansion of the number of short tandem repeats within or in the vicinity of a gene can affect gene expression
• Repeats can predispose to large deletions and duplications

Question 30

What are larger deletions and insertions?

Answer 30

• usually caused by unequal crossing-over between repeat sequences.
• may affect a gene, several genes or section of a chromosome.
• clinical effects depend on genes involved and gene dosage.

Question 31

How does the structure of the genome form the basis of genetic diseases?

Answer 31

Variations/mutations may involve
– a single gene (Mendelian inheritance)
– a chromosomal segment (or whole chromosome) and so affect thousands of genes
(copy number variation)
– several variants of genes acting with environmental influences (multifactorial inheritance)

Question 32

Summary

Answer 32

• DNA replication and repair processes fail only rarely
• A permanent change in DNA is a mutation (in classical terminology)
• A protein with an altered amino acid sequence may have no or an altered function
• Mutations occurring in germ cells can be passed on to offspring
• Mutations occurring in somatic (i.e. not germ) cells are not transmitted to offspring

Question 33

Summary of the effects of DNA sequence variants

Answer 33

• Most are polymorphic variants (SNPs) with no known effect on phenotype
• Within an exon may have no effect on phenotype if there is no change in amino acid
• Changing an amino acid (missense substitution)
– may cause clinical effects if protein function altered
– or have no phenotypic effect if there is no effect on protein function
• Detection of a pathogenic mutation allows genetic testing for other family members and in some diseases influences clinical management