basics of mutations and polymorphisms

acquired mutations

generate new alleles

not inherited

DNA damage - alteration in the chemical structure of the DNA leading to mutations/single nucleotide polymorphisms:

• environmental agents

• by-products of normal metabolism

• spontaneous damage

environmental agents

UV and ionising radiation

causes DNA strand breakage, single or double

breaks → mis-pairing → mutations/chromosome aberrations

radiation DNA damage

indirectly - water can readily absorb a large amount of radiation becoming ionised forming higher reactive DNA damaging free radicals e.g. hydroxyl ROS characterised by an unpaired electron

directly - radiation can collide directly with the DNA itself which causes ionisation

UV light

UVB component (280-320nm) is highly mutagenic

UV on prolonged exposure gets absorbed by pyrimidine bases forming dimerisation: cyclobutane pyrimidine dimers and pyrimidine pyrimidone photoproducts

cyclobutane pyrimidine dimers account for around 85% of primary lesions in UV-irradiated DNA

what do UV dimers target?

protooncogenes and tumour suppressor genes

e.g. p53 which usually plays a role responding to damage induced by UV - causes cell cycle arrest allowing time for DNA repair of UV photoproducts or initiation of apoptosis - mutations lead to lose of control of these functions so damaged cells are left unchecked and replicate

natural or man-made genotoxic chemicals

single-stranded breaks, base substitutions, base insertions, base deletions

e.g. benzo(a)pyrene - natural carcinogen found in fossil fuels and formed by incomplete combustion - the body attempts to metabolise beno(a)pyrene via CYP1A1, forming the metabolite benzo(a)pyrene diol epoxide (BPDE) which can bind to DNA predominantly forming covalent adducts at the N2 position of guanine, highly carcinogenic → high-fidelity DNA polymerases stall at the lesion site as they cant accommodate the bulky adduct → base misincorporation → dysfunctional protein such as p53

by-products of normal cellular metabolism - endogenous ROS

endogenous ROS e.g. hydroxyl radicals → reacts with bases or the backbone of DNA, leading to strand breaks

ROS are generated in mitochondrial ETC (oxidative phosphorylation - aerobic respiration) normally the ETC transfers electrons through complexes I-IV to reduce oxygen to water, however sometimes electrons leak prematurely and partially reduce oxygen - forming superoxide ROS → hydroxyl radical → oxidative stress

this can be controlled by detoxification, however some ROS can escape detoxification and are free to cause cellular damage

why are mitochondria susceptible to ROS attack?

close to the inner mitochondrial membrane where ETC occurs and free radicals are found

lack protective proteins such as histones, therefore highly exposed to free radical attack

free radical-induced mt DNA damage/mutations

ultimately lead to a less efficient ETC - mitochondria gradually deteriorate and there is a steady rise in ROS generation → decrease in vital ATP production → energy deprivation

mitochondrial dysfunction is a characteristic of aging and chronic diseases e.g. late-onset neurodegenerative disorders and CV diseases - all involve muscle cell contraction and nerve cell firing which depend on ATP

by-products of normal cellular metabolism - exogenous ROS

exogenous ROS are generated environmentally by: ionising radiation, pollutants, tobacco, smoke, drugs, xenobiotics

environmental stress ROS increase → DNA damage, diversity of lesions such as ss breaks, crosslinking of bases, base modfications, and abasic sites

spontaneous or intrinsic DNA damage

damage that occurs independently of environmental factors

such as errors in replication - normally controlled by DNA repair mechanisms and lesions are left unchecked: base pair mismatches, ss breaks, insertionsm deletions, and depurination/deamination

mutation

a mutation is any change in a DNA sequence in an allele that changes this normal allele to a rare and abnormal variant - it has to be present in less than 1% of the population

polymorphism

the rare allele must have a frequency of at least 1% or more of the population

point mutations, single base pair or nucleotide polymorphisms (SNPs)

small scaled mutations which cause changes in the genetic sequence - when one single codon in a gene is altered, when translated, may significantly impact on protein production

• base substitutions

• insertions

• deletions

base substitutions

one base is substituted by a different base:

transitions: swap a purine with a purine or pyrimidine with pyrimidine

transversions: swap a purine with a pyrimidine or vice versa e.g. G to T in p53 gene - when G base is damaged from benzo(a)pyrene adduct formation which is usually corrected with excision repair → DNA polymerase will often insert an adenine opposite to the damaged guanine → when replicated, T is incorporated opposite the A base → dysfunctional p53 protein

types of base substitutions

missense

nonsense

silent

missense mutations

point mutation results in a codon for a different amino acid and can result in a non-functional protein

e.g. sickle cell anaemia - GAG → GTG, codes for valine which causes haemoglobin to be less soluble under low oxygen which distorts it

nonsense mutations

point mutation in a sequence of DNA that results in a premature stop codon in the transcribes mRNA → forms truncated, incomplete, and usually non-functional protein

15-30% of all inherited diseases are due to nonsense mutations e.g. cystic fibrosis, Duchenne muscular dystrophy

silent mutations

point mutations in DNA that does not significantly alter the pheotype of that organism - can occur in non-coding regions, but may occur within exons

insertions

occurs when one or more base pairs are added to the sequence - increases length of DNA

frameshift mutation - if it occurs in an exon region, it will completely alter codon readout and translated protein - protein is usually truncated and non-functional

Tay Sachs

Tay Sachs - autosomal recessive and fatal disease of the nervous system

insertion of 4 base pairs in exon 11 of the hexosaminidase gene on chromosome 15 → frameshift mutation and hexosaminidase A deficiency (enzyme crucial to the CNS)

Gilberet’s syndrome

common genetically inherited disorder, faulty UGT1A1 gene

normally causes conjugation of bilirubin with lipophilic molecules - if mutated, causes the liver to have problem in removing bilirubin from the blood → build up of bilirubin in the bloodstream, giving a yellow tinge of the skin and eyes

dinucleotide insertion into the TATA box region of the bilirubin UGT1A1 gene promotor → decrease in hepatic enzyme activity

deletions

frameshift mutation where base pairs are removed from the DNA sequence - greatly vary in size (single base or whole gene)

e.g. cytosine in codon 39 in beta-globin gene is deleted - following reading frame is altered and continues until a stop codon is encountered → beta thalassemia (blood disease)

chromosomal mutations and abnormalities

large scale deletion: section or whole chromosome can be deleted e.g. Jacobsen syndrome - rare congetial disorder ruslting from a deletion in chromosome 11

large scale insertion: portion of one chromosome is deleted from one site and is inserted into another e.g. haemophilia A - blood clotting is prevented due to production of dysfunctional blood clotting factor VIII enzyme

inversions: reversing the orientation of a chromosomal segment - occurs when a single chromosome undergoes breakage and rearrangement within itself (factor VIII gene in haemophilia A) → dysfunctional proteins

translocations

interchange of genetic material from different chromosomes → gene fusion

may play important role in tumorigenesis as fusion genes tend to produce much more active abnormal protein than non-fusion genes → uncontrolled downstream signalling and growth

e.g. chromic myelogenous leukaemia (CML) - extention in chromosome 9 and shortening of chrmosome 22 → BCR-ABL (Philadelphia translocation)

gene amplification

instead of making a single copy of a region of a chromosome, many copies are produced → overproduction of the mRNA and protein

plays important role in the development and progression of 20% of certain aggressive types of breast cancer

over amplification of the RTK erbB2 (HER2) gene on chromosome 17 - erbB2 become an important biomarker and target for therapy in breast cancer patients (trastuzumab)

beneficial mutation variants

• lactose tolerance - evolutionary, natural selection

  • used to lack the ability to digest milk as adults - lactase gene was suppressed after weaning

  • 1/3 of the world can process milk as adults

  • lactase mRNA production is regulated by transcription factors and activators - bind to DNA enhancers sites upstream of the lactase gene transcription site e.g. OCT-1 enhancer - C to T SNP mutation increases its binding affinity for the OCT-1 transcription activator protein → increases promotor binding to the transcription complex → increase lactase expression

• sickle cells

  • sickle cell anaemia requires two copies of the mutated gene

  • mosquitoes that carry the parasite for malaria require human blood cells to complete its life cycle → those with sickle cell anaemia can prove beneficial offering a selective advantage is a person only has one copy of the mutated gene