Chapter 12 Mutations and Mutagenesis

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Chapter 12Mutations and Mutagenesis
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GENE MUTATIONSA mutation is a detectable and
heritable change in genetic material that is not
caused by genetic recombination.
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Mutations can occur in various parts of a gene

• Coding sequence (exons)
• Introns
• Transcription control regions (i.e. promoter)
• Mutations in exons introns can affect amino
  acid sequence
• Mutations in control regions generally affect
  gene expression levels

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Not all mutations are harmful!

• Mutation is necessary to provide a variable
  background on which selective forces can act
• Many examples of beneficial mutations are known
• Lactase absorbers have autosomal dominant
  mutation allowing for production of lactase
  through adulthood
• Mutation in cell surface protein (CCR5) confers
  resistance to HIV infection

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Somatic mutations

• Occur in non-germline (somatic) cells
• Mutation occurs during DNA replication preceeding
  mitosis
• Affects subsequent somatic cell descendants only
• Responsible for some cancers

Germline mutations

• Occur in germline cells (precursors to egg
  sperm)
• Mutation occurs during DNA replication preceeding
  meiosis
• Possibility of perpetuation through transmission
  to offspring

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Types of mutations

• Point mutations
• Repeat expansions
• Pseudogenes, transposable elements, and
  chromosomal aberrations

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Point mutations substitutions

• a change of a single nucleotide to one of the
  other three possible nucleotides
• Transition
• purine replaces purine A -gt G or G -gt A
• OR pyrimidine replaces pyrimidine C -gt T or T
  -gt C
• Transversion
• purine replaces pyrimidine A or G -gt T or C
• OR pyrimidine replaces purine T or C -gt A or G
• Transitions are tolerated better and are more
  common

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Silent mutations

• Point mutation that does not change meaning of
  codon
• Due to degeneracy of genetic code
• Silent mutations do not result in changes in
  polypeptide sequence or function

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Missense mutations

• A point mutation that exchanges one codon for
  another causing substitution of an amino acid
• Missense mutations may affect protein function
  severely, mildly, or not at all.
• HbS mutation
• glutamic acid -gt valine causes sickle cell anemia

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Nonsense mutations

• A point mutation changing a codon for an amino
  acid into a stop codon (UAA, UAG or UGA).
• Premature stop codons create truncated proteins.
• Truncated proteins are often nonfunctional.
• Some truncations have dominant effects due to
  interference with normal functions.
• Factor XI deficiency
• Blood clotting disorder
• most common cause is a nonsense mutation
• changes glutamic acid -gt stop codon
• Short protein cannot function in clotting.

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Not all point mutations impact protein function

• Silent mutations do not alter the amino acid
  encoded.
• Example AAA and AAG both encode the amino acid
  lysine
• DNA sequence altered but protein sequence remains
  unchanged.
• These changes are called synonymous mutations

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Not all point mutations impact protein function

• Missense mutations alter the encoded amino acid
  to another amino acid
• These changes are called nonsynonymous mutations
• Some nonsynonymous mutations are conservative
  (or neutral) replacement with chemically
  similar amino acid may not alter function
• The impact of a missense mutation is not
  predictable from protein sequence alone

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Point mutationsInsertion or deletion mutations

• The genetic code is read in triplet nucleotides
  during translation.
• Addition or subtraction of nucleotides not in
  multiples of three leads to a change in the
  reading frame used for translation. Amino acids
  after that point are different, a phenomenon
  called a frameshift.
• Addition or subtraction of nucleotides in
  multiples of three leads to addition or
  subtraction of entire amino acids but not a
  change in the reading frame.

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Insertion or deletion mutations

• Deletion is the removal of sequences
• 2/3 of Duchenne muscular dystrophy cases are
  caused by large deletions
• Many cases of male infertility caused by small
  deletions in Y chromosome
• Insertion is the addition of sequences
• Gaucher disease can be caused by a single base
  insertion creating
• frameshift prevents production of an enzyme
  needed to break down glycolipids in lysosomes
• Buildup of glycolipids enlarges liver spleen,
  causes neurological damage
• Disease common among Jews of eastern European
  descent

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Insertion mutations

• May repeat part or all of gene sequence
• Tandem duplication 2 copies of gene next to each
  other
• Example Charcot-Marie-Tooth disease
• Characterized by numb hands and feet
• Caused by a tandem duplication of 1.5 million
  bases

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Point mutations may not be in reading frame at
all!Example Becker muscular dystrophy

• Adult, milder form of muscular dystrophy
• In 15 of cases, mutation in promoter of
  dystrophin gene
• Transcription rate slowed (ltmRNA)
• Protein is normal, but not as much produced
• Muscle function suffers
• In other 85 of cases, dystrophin protein is
  truncated non-functional (mutations in reading
  frame)

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Expanding triplet repeats

• Some genes are particularly prone to expansion of
  repeats.
• Number of repeats correlates with earlier onset
  and more severe phenotype.
• Expansion of the triplet repeat and coincident
  increase in severity of phenotype occur with
  subsequent generations, a phenomenon termed
  anticipation.

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Triplet repeat disorders
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Fragile X syndrome

• Most well-studied of triplet repeat expansion
  disorders
• Caused by disruption of FMR1 (fragile X mental
  retardation 1) on X chromosome
• Protein FMRP (from FMR1 gene) binds mRNAs in
  brain cells affects expression of genes
  essential for brain development
• CCG repeat in 5UTR of FMR1 normally repeated
  6-50X (avg 29 copies)
• 50-230X repeats termed fragile X premutation
• Rarely results in symptoms
• Offspring have increased risk of Fragile X
  syndrome
• Significantly more expansion (e.g. 700-1000X)
  Fragile X syndrome
• Bases in repeat region and promoter become highly
  methylated
• Transcription inhibited
• Ribosomes do not efficiently translate the few
  mRNAs that are produced
• Quantity of FMRP produced is inadequate brain
  development inhibited
• Also (rarely) caused by missense or deletion
  mutations

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Myotonic dystrophy a triplet repeat disease

• 5 -37 copies of CTG repeat normal phenotype
• 50-1000 repeats myotonic dystrophy
• Genes with 40 copies are unstable and can gain
  (or less commonly lose) repeat copies in
  successive generations.

Anticipation!!!!
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Mutation overview.......
Example
Type of mutation
Expanding mutation
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Different mutations may cause the same disorder
Mutations in the LDL receptor disrupt function
leading to increased blood cholesterol and early
heart disease (Familial hypercholestrolemia)
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Not all mutations impact protein function

• Conditional mutations are those that only produce
  a phenotype under particular conditions or
  environments
• G6PD
• enzyme in immature RBC used to extract energy
  from glucose
• Necessary in response to oxidants, chemicals that
  strip electrons from other molecules
• X-linked gene
• gt1 million people worldwide with G6PD deficiency
• High levels of oxidants occur when eating fava
  beans, inhaling certain pollens, taking certain
  antimalarial drugs (primaquine)
• Conditions Individuals with mutations in
  G6PD
• Low oxidants no phenotype
• High oxidants red blood cells burst, anemia

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Pseudogenes

• A pseudogene is a DNA sequence reminiscent of a
  gene but which is not translated (may or may not
  be transcribed).
• Pseudogenes may have evolved from original
  functional gene by duplication and acquired
  mutation(s).
• Crossing over between a pseudogene and an intact
  gene can disrupt gene expression.
• Gaucher disease
• Pseudogene intact gene share 95 sequence
  similarity
• Separated by 16kb
• Some cases of disease result from crossover
  between intact gene and pseudogene
• Result fusion gene (part intact gene, part
  pseudogene)
• Functional protein cannot be translated from
  fusion gene

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Transposable elements(transposons or jumping
genes)

• Can disrupt site from which they jump
• Can shut off transcription of gene into which
  they jump
• Can alter reading frame of gene into which they
  jump
• Genes interrupted by transposable elements are
  generally nonfunctional
• One method of creating pseudogenes

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Chromosomal aberrations

• Can eliminate, duplicate, or rearrange
  chromosomes
• Include 100s to 1,000s of bp
• Will be topic of future lecture!

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Causes of mutation

• Spontaneous vs.
• Induced

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Spontaneous Mutations

• De novo or new mutations
• Caused by errors in DNA replication, NOT exposure
  to known mutagens
• Considered natural, NOT "man made
• Represent the low level of genetic changes that
  occur through time
• Has lead to the idea of the genetic clock

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Spontaneous mutation

• DNA bases exist in two different tautomeric forms
• For short periods, DNA bases are in unstable
  tautomeric form
• If unstable DNA base is inserted during
  replication, mismatch can occur
• Mismatched base passed along as mutation into
  daughter cells

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Spontaneous mutation rate

• Rate differs for different genes
• Size dependence
• (large gene more chances for mutation)
• Sequence dependence
• Hot spots
• On average 1 in 100,000 chance of acquiring a
  mutation in a gene each round of replication
• Each individual has multiple new mutations.
  Most, by chance, are not in coding regions of
  genes

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Determining mutation rate

• Estimates of spontaneous mutation rate can be
  derived from observation of dominant traits
• For autosomal genes
• mutation rate number of cases
• 2 ( of
  individuals)
• dominant trait only

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Mutation rates of genes causing disease
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Mutations in pathogens

• Bacteria and viruses have short generation time,
  so mutations are more frequent
• BACTERIA
• Mutations can lead to antibiotic resistance
• Overuse and incomplete course of treatment
  increases chances of antibiotic resistance
  arising
• VIRUSES
• Influenza vaccines reassessed each season to
  accommodate viral changes
• Rapid mutation of HIV virus makes treatment
  difficult

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Mutational hot spots exist

• Region of gene where mutations occur at higher
  than usual frequency
• Often occur in short repetitive sequences
• Replication machinery confused by
  repetitiveness?
• Pairing of repeats in same strand may interfere
  with replication or repair enzymes
• Example
• gt1/3 of mutations causing alkaptonuria occur at
  or near one or more CCC repeats
• BUT CCC repeats only account for 9 of gene!
• CCC repeats are mutational HOT SPOTS

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Repetitive and symmetrical DNA sequences may
confuse replication machinery
Replication machinery has harder time reading
repetitive sequences
Inverted repeats cause hairpins that disrupt
replication
Palindromes disrupt replication, causing small
insertions or deletions
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Gene duplication can result in mispairing during
meiosis

• Normally, 2 a-globin genes present on each
  chromosome 16 (4 genes total)
• Misalignment during meiosis I can result in
  gametes with 1 (deletion) or 3 (insertion) genes
  per chromosome
• Fertilization with normal gamete results in
  individual with 3 or 5 a-globin genes
• a-thalassemia (contrast to ß-thalassemia)
• 3 a-globin genes normal
• 2 a-globin genes mild anemia, tire easily
• 1 a-globin gene severe anemia
• Carriers have heterozygous advantage
• protected from Plasmodium falciparum (malaria)

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Induced mutations Some definitions

• Mutagen
• Agent which can cause mutations
• Carcinogen
• Agent which cause cancer
• Clastogen
• Agent that causes chromosome abnormalities
• Teratogen
• Agent that increases incidence of congenital
  malformations

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Two major types of induced mutations Radiation
and Chemical
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Types of RadiationIonizing Radiaton

• Sufficient energy to remove electrons from atoms
  create free radicals
• Exist naturally and can be produced by humans
• Breaks sugar-phosphate backbone of DNA
• Three types
• Alpha radiation
• mostly absorbed by skin
• Examples uranium and radium
• Tends to not harm health, but can do damage if
  inhaled or ingested
• Beta radiation
• Can penetrate into body farther than alpha
  radiation
• Examples tritium (hydrogen isotope), Carbon-14,
  Strontium-70
• Tends to not harm health, but can do damage if
  inhaled or ingested
• Gamma rays
• Penetrate through entire body
• Damages tissues
• Examples plutonium and cesium isotopes
• Form used intentionally to kill cancer cells
• X rays

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Types of RadiationNon-ionizing radiation

• Not enough energy to strip electrons from atoms
• Electrons temporarily assume higher energy levels
• UV rays (ultraviolet light)
• commonly found in sunlight
• Penetrates only uppermost layers of cells
• Readily absorbed by DNA
• C and T particularly susceptible to excitation
  and damage
• causes thymine-thymine dimers, leading to
  mutations in DNA on opposite (synthesized) strand

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Relax! Most exposure to radiation is natural
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Chemical mutagens

• Base analogs
• Nitrogenous bases similar to DNA bases
• Can be incorported into DNA during replication
• Pairing propertied differ, so newly synthesized
  strand has mismatch
• 5-bromouracil
• Analog of T (incorporated in place of T)
• In more stable enol form of 5-BU, pairing occurs
  with G (A-gtG transition)
• 2-amino purine
• Analog of A (incorporated in place of A)
• May pair with T or C
• When paired with C, T-gtC transition occurs

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Base analog 5-bromouracil
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Chemical mutagens (continued)

• Intercalating agents
• Intercalate between stacked DNA bases
• Cause insertion/deletion mutations -gt can lead to
  frameshift
• Example acridine dyes (acridine orange
  proflaven), ethidium bromide, ICR compounds
• Deaminating agents
• Removes amine (NH2) groups from C (-gt U), A
  (-gthypoxanthine), or G (-gt xanthine) and can
  cause mispairing
• Examples bisulfite compounds, nitrous acid
• Hydroxylating agents
• Add hydroxyl groups to nucleotides bases
• Example hydroxylamine adds hydroxyl group to
  amino group of C (-gt hydroxyaminocytosine)
• Pairs with A (G-gtA transition)
• Alkylating agents
• Add alkyl (methyl or ethyl) groups to nitrogenous
  bases
• May cause transitions (most common),
  transversions, insertions, deletions, and
  chromosome breaks
• Example polycyclic aromatic hydrocarbons (many
  types of PAH found in tobacco smoke)

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Where can you find mutagens?
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Detecting mutationAmes test

• An in vitro test of the mutagenicity of a
  substance using Salmonella bacteria with mutation
  in gene for histidine (i.e. no His produced)

• Bacteria exposed to test substance.
• Growth of bacteria on media without histidine
  recorded.
• Bacteria only grow if reversion mutations have
  occurred.
• Rate of mutation is determined.
• Substance can be mixed with mammalian liver
  tissue prior to testing to mimic toxin
  metabolizing in humans.
• 90 correlation between mutagenicity
  carcinogenicity

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Hemophilia in Royalty
Detecting mutation pedigree analysis
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DNA Repair

• Photoreactivation
• Nucleotide excision repair
• Base excision repair
• Mismatch repair
• Uracil mutation repair
• Alkylation mutation repair

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DNA replication is very accurate

• 1 in 1 million bases incorporated incorrectly
• DNA polymerase has proofreading ability in
  eukaryotes
• 3-5 exonuclease
• First line of defense against mutation
  accumulation
• Proofreading by other enzymes in prokaryotes (not
  DNAP)

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Not all organisms can repair DNA equally well

• mtDNA has no repair mechanisms
• High intrinsic mutation rate
• Deinococcus radiodurans is worlds toughest
  bacterium
• 1956 discovery in can of spoiled ground meat
• Had withstood radiation used to sterilize the
  food
• Can tolerate 1,000x radiation that a person can
• Can live in nuclear reactor!

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DNA Repair

• Errors in DNA replication or damage to DNA create
  mutations.
• Most errors and damage are repaired by the cell.
  
• The manner in which DNA repair occurs depends
  upon the type of damage or error.

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Photoreactivation (light repair)

• Action of the enzyme photolyase
• - Requires light energy (320-370 nm, blue light)
• - Does not remove any nucleotides
• - Repairs dimer formation by splitting the TT
  bonds.
• Found in many modern species, but not humans

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Excision repair (dark repair)

• Bond cut between DNA sugar and base
• Offending base(s) is/are cut out
• DNA polymerase fills in correct nucleotides
• 2 types in humans
• Nucleotide excision repair
• Base excision repair

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Nucleotide Excision Repair

• UvrABC endonuclease
• 1 cut 8 nucleotides on the 5' side of the damage.
• 1 cut 4 nucleotides on the 3' side of the damage.
• Up to 30 bp damaged DNA are removed.
• Gap refilled by DNA Pol I
• Ends joined by DNA ligase
• Used to repair bases damaged due to
• Chemical carcinogens
• UVB (thymine-thymine dimers)
• Oxidative damage

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Nucleotide Excision Repair
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Base Excision Repair

• Replaces up to 5 nucleotides
• Specific for errors resulting from oxidative
  damage due to oxygen free radicals
• Oxygen free radicals produced during chemical
  reactions (e.g. metabolism, transcription)
• Free radicals damage DNA

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Mismatch repair

• Corrects replication errors
• Enzymes proofread newly replicated DNA for
  bubbles
• bubbles represent nucleotides that are not
  paired correctly
• The incorrect base is excised and replaced

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Uracil DNA Glycosylase Repair

• Removes Uracil from DNA
• Cleaves the glycosidic bond (between the base and
  the sugar) and not the phosphodiester bond
• AP endonuclease recognizes the hole and removes
  the rest of the nucleotide
• Gap is filled in by DNA polymerase I

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Alkylation Mutation Repair

• Mutations that result from additional methyl or
  ethyl groups added to nucleotides.
• Repaired by the action of O6 methylguanine
  methyltransferase.

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Failure of DNA repair

• When DNA repair fails, fewer mutations are
  corrected leading to an increase in the number of
  mutations in the genome.
• The protein p53 monitors repair of damaged DNA.
• If damage is too severe, the p53 protein
  promotes programmed cell death or apoptosis.
• Mutations in genes encoding DNA repair proteins
  can be inherited and lead to overall increase in
  mutations when DNA errors or damage are no longer
  fixed efficiently.

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DNA replication and repair disorders
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Human Diseases resulting from mutations in DNA
repairXeroderma Pigmentosum

• Autosomal recessive disease
• Deficiency in repair of thymine dimers
• May involve mutations is at least 6 different
  genes
• Affected individuals gt10,000x risk of skin
  cancers compared to unaffected individuals
• gt1/2 develop cancer before they are teenagers
• Affected individuals must remain indoors
• Brief exposure to sunlight causes painful
  blisters
• Some individuals have neurological abnormalities
  in addition to skin cancers.

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Xeroderma Pigmentosum Phenotype
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Human Diseases resulting from mutations in DNA
repair

• Fanconi's Anemia
• Impaired removal of DNA interstrand cross-links
  such as those caused by the antibiotic
  mitomycin-C.
• Bloom's Syndrome
• High frequency of chromosome breakage.
• Ataxia-telangiectasis
• Defect in kinase that functions as cell cycle
  checkpoint
• Cells do not pause to repair mispaired bases
• Extremely high rates of cancers, esp. of blood
• Poor balance/coordination (ataxia), red marks on
  face (telangiectasia), delayed sexual maturation,
  high risk of lung infection diabetes mellitus
• Heterozygotes have sensitivity to ionizing
  radiation
• All of the above cause the affected individual to
  be prone to malignancies resulting from problems
  in DNA repair processes.