Gene and chromosomal mutations

Mutations

• although occasional genetic changes enhance the long-term survival of a species, the survival of an individual demands a high degree of genetic stability

• when the cell’s DNA maintenance processes fail —> mutation

• Definition of mutation: permanent change in DNA, which is an alteration to an organism’s characteristics that is inherited due to a change in the genetic material of a cell

• it can destroy an organism if it occurs in a vital position in the DNA sequence

• mutations can involve

  • just a single or a few nucleotide pairs

  • large regions of a chromosome

• types of mutations in multicellular organisms

1. Germline mutation

• mutation occurs in germline cells (consisting of germ cells and gametes) - it may be transmitted to the offspring and to successive generations (can be inherited)

• if the mutation has an adverse effect on the phenotype of an organism, the mutant condition is referred to as a genetic disorder, or hereditary disease

2. Somatic mutation

• mutation occurs in somatic cells

• these mutations are not inherited by the progeny and hence not passed on to the next generation

Gene/point mutations

• Gene/point mutations involve chemical changes that affect the DNA sequence of just one gene

• they involve changes at specific sites in a gene - result in change in one or a few bases in the DNA sequence

• in gene/point mutation, 2 basic types of changes to a gene can occur

1. Nucleotide substitutions

• replacement of one nucleotide pair with another, resulting in one of the following

a. missense mutation: nucleotide substitution in a DNA sequence results in the translation of a different amino acid

b. nonsense mutation: nucleotide substitution in a DNA sequence results in a codon for an amino acid being changed into a stop codon, leading to premature termination of translation

c. silent mutation: nucleotide substitution in a DNA sequence changes the mRNA codon. however, the same amino acid is inserted into the protein because of the degeneracy of the genetic code

d. neutral mutation: nucleotide substitution in a DNA sequence changes the mRNA codon and amino acid translated. however, the resulting amino acid substitution produces no detectable change in the function of the protein translated.

note: these are 4 examples of mutation, but they are NOT TYPES of mutations

2. Nucleotide insertions or deletions

• addition or deletion of one or more nucleotide pair

• depending on the location in the DNA sequence and the number of nucleotide pairs added or deleted, one of the following will ensure

a. Addition or deletion of deoxynucleotides in multiples of 3 results in:

• missense mutation - mRNA codon was added or deleted and the resulting polypeptide has an amino acid added or deleted respectively

OR

• nonsense mutation - stop codon was added, leading to premature termination of translation

b. Addition or deletion of deoxynucleotides not in multiples of 3 results in:

• frameshift mutation - mRNA codons subsequent to the insertion or deletion are changed, resulting in

  • extensive missense mutation - subsequent amino acid sequence of the polypeptide is changed OR

  • nonsense mutation - codon for an amino acid is changed to a stop codon, resulting in a truncated protein

Effects of nucleotide substitutions - Missense mutation

• nucleotide substitutions may or may not affect the amino acid sequence of a protein depending on the location of the substitution

Missense mutation

• nucleotide substitution in DNA sequence changes the mRNA codon, resulting in the translation of a different amino acid

• amino acid sequence of the polypeptide is changed = resulting in a change in the specific 3-dimensional conformation of the protein = function of the protein is altered

*change in a.a —> change in a.a sequence —> change in 3D conformation —> change in protein function

e.g. sickle cell anaemia

Effects of nucleotide substitutions - nonsense mutation

• nucleotide substitution in a DNA sequence changes a codon for an amino acid into a stop codon

= premature termination of translation

= resulting polypeptide will be truncated (shorter) than normal polypeptide encoded

• amino acid sequence of the polypeptide is shortened

= change in the specific three-dimensional conformation the protein

= function of the protein is altered

• nearly all nonsense mutations result in non-functional proteins (due to truncated polypeptides)

Effects of nucleotide substitution - Silent mutation

• nucleotide substitution in a DNA sequence changes the mRNA codon

• however, the same amino acid is inserted into the polypeptide because of the degeneracy of the genetic code

• the amino acid sequence of the polypeptide is unchanged

• = resulting in no change in the specific three-dimensional conformation of the protein

= no change in the function of the protein

Effects of nucleotide substitution - Neutral mutation

• nucleotide substitution in a DNA sequence changes the mRNA codon

• however, the resulting amino acid produces no detectable change in the function of the protein translated

• this could arise from

1. the substitution of the original amino acid with an amino acid of similar physical and chemical properties OR

2. the substitution of an amino acid residue that is non-essential to that protein’s structure and function

• the amino acid sequence of the polypeptide is changed, BUT there is no change in the overall three-dimensional conformation of the protein and hence the function of the protein is not altered

Effects of nucleotide insertions or deletions

• nucleotide insertions or deletions are additions or losses, respectively, of one or more nucleotide pairs in a gene

• often has deleterious effects

• resulting mRNA is read as a series of non-overlapping codons, an insertion or deletion of nucleotides NOT in multiples of 3 will result in frameshift mutation (reading frame of the mRNA is changed)

  • all the nucleotides downstream of the insertion/deletion site will be improperly grouped into codons, resulting in extensive missense

  • frameshift may also cause a new, premature stop codon to be generated (nonsense mutation) in the reading frame, or result in a read-through of the normal stop codon, resulting polypeptides of altered lengths

  • a frameshift usually results in a non-functional protein

Effects of nucleotide insertion/deletion - Deletion mutation

• single base deletion mutation leads to frameshift

• e.g. in figure: deletion of A in the DNA sequence results in the alteration of subsequent codons in the corresponding mRNA, hence different amino acids are coded for subsequently

= incorrect amino acid sequence of the polypeptide chain, and consequently incorrect three-dimensional protein conformation and function

Effects of nucleotide insertion/deletion - Insertion mutation

• single base insertion mutation leads to frameshift

• e.g. in figure: insertion of A in the DNA sequence results in the alteration of subsequent codons in corresponding mRNA

= different amino acids are coded for subsequently

= incorrect amino acid sequence of the polypeptide chain, and consequently incorrect three-dimensional protein conformation and function

Effects of nucleotide insertion/deletion - Insertion or deletion mutation (in multiples of three)

• e.g. insertion of 2 sets of nucleotides in multiples of three does not lead to frameshift

• amino acid sequence contains 2 additional glutamines —> 3D conformation of the protein may be changed hence leading to a change in the functions of the protein

Summary - point mutation leading to different consequences

Summary table (2)

Case study: Gene mutations resulting in phenotypic change

—> Sickle-cell anaemia (how gene mutations can result in disease)

• involves a mutation in the β-globin gene, which encodes one of the polypeptide subunits that make up haemoglobin (Hb) (Hb - 2α and 2β subunits)

Genetic and molecular basis

• substitution of a thymine for an adenine at one position in the Hb gene (template strand), which results in a missense mutation

T —> A

• 6th amino acid residue in polypeptide is changed from a glutamate (hydrophilic) to a valine (hydrophobic)

• change in property of amino acid —> change in the interactions/bonds formed with neighbouring a.a

= specific three-dimensional conformation and function of the Hb protein is altered

• this substitution creates a hydrophobic spot on the outside of the Hb protein that sticks to the hydrophobic region of an adjacent Hb protein’s beta chain

• mutant Hb subunits tend to stick to one another when the oxygen concentration is low, particularly when the red blood cells are in capillaries and veins

• the aggregated proteins form fibre-like structures within red blood cells

• at high oxygen concentration, haemoglobin resumes globular haemoglobin structure

Physiological effects

• the fibre-like structures cause the RBC to lose their normal morphology and become sickle-shaped

• sickles cells are less able to move through capillaries and can block blood flow, resulting in severe pain and cell death of the surrounding tissue due to shortage in oxygen

• the sickles RBC are also fragile and easily destroyed, further decreasing the oxygen carrying capacity of the blood

*point mutation (nucleotide substitution) —> change in a.a sequence —> change in protein structure (3D conformation) —> change in protein function (decreased capacity to carry blood cos of the change in morphology of RBC) —> disease

Figure showing missense mutation causing sickle-cell anaemia

Causes of gene mutations

• mutagenesis: production of mutations

• 2 groups of causes

1. Spontaneous mutations

• mutations that occur naturally - without the use of chemical or physical mutagenic agents

• result of errors that occur during DNA replication, recombination or repair (can lead to both gene and chromosomal mutations)

• DNA replication and repair

  • mistakes: although DNA replicates with high fidelity, DNA polymerase sometimes inserts the wrong nucleotide/too many/too few nucleotides into DNA sequence

  • DNA polymerase make mistakes at a rate of about 1 in every 100 000 nucleotides (~120 000 mistakes every time a cell divides with 6 billion bp in each human diploid cell)

  • attempts at correction: proofreading, where some of the mistakes are corrected immediately; some are corrected after replication during mismatch repair

  • during proofreading, DNA polymerase enzymes recognise mistakes and replace the incorrectly inserted nucleotide so that replication can continue

  • after replication, mismatch repair reduces the final error rate even further

—> incorrectly paired nucleotides cause deformities in the secondary structure of the final DNA molecule - during mismatch repair, enzymes recognise and fix these deformities by removing the incorrectly paired nucleotide and replacing it with the correct nucleotide

• some replication errors fail to be recognised by the repair enzymes = these altered nucleotide sequences can be passed down from one cellular generation to the next

• if they occur in cells that give rise to gametes, can be transmitted to subsequent generations of the organism

2. DNA slippage

• daughter or parental DNA strand slips during DNA replication followed by folding back of the strand

• mispairing between the daughter DNA strand and the parental template strand

• causes parts of the DNA which are folded back to be copied more than once

• if this duplicated DNA segment corresponds to a gene, it will result in gene duplication

3. Induced mutations

• result of deliberate application of mutagens - chemical or physical agents - that result in increased mutation rates

Figure of DNA slippage

Mutagens and effect on DNA

Physical agents (radiation)

1. X-rays

• results in the production of free radicals of water (the OH radical) which are chemically very reactive

• free radicals interact with DNA to produce double-stranded breaks leading to chromosomal rearrangements and deletions (KIV: Pg 18 on Chromosomal Mutations)

2. Ultraviolet (UV) rays

• are absorbed by bases of DNA. This may result in

  • the production of a covalent attachment between adjacent pyrimidines in one strand, usually thymine dimers, or

  • base pair substitutions, insertions and deletions

• DNA replication and transcription are blocked – this is lethal if unrepaired

Chemical agents

1. Base analogues

• are molecular structures that are similar to the bases normally found in DNA

• may be incorporated into DNA in place of the normal bases during DNA

replication, hence producing base substitutions

E.g. Bromouracil (5-BU), which

o can exist in alternate states

o each of which can pair with a different base in DNA, hence producing base substitutions

2. Base-modifying agents

• modify the chemical structure and properties of bases

• leads to mispairing during DNA replication and hence base substitution

E.g. Alkylating agents such as ethylmethylsulfonate (EMS) and mustard gas.

o transfer alkyl groups to bases, modifying their chemical structure

3. Intercalating agents

• are flat molecules with multiple ring structures

• insert themselves (intercalate) between adjacent bases in one or both strands of

the DNA helix

• leads to insertions or deletions during DNA synthesis and hence frameshift

mutations

E.g. Proflavin, acridine, ethidium bromide.

Chromosomal mutation/aberration

Definition:

1. a change in the structure of a chromosome (involving several gene loci) OR

2. a change in the number of chromosomes

Change in chromosome structure

• structural changes that delete, add or rearrange substantial portions of one or more chromosomes

• deletion and duplication of genes or part of a chromosome and rearrangements of genetic material in which a chromosome segment is inverted, exchanged with a segment of non-homologous chromosome, or merely transferred to another chromosome - translocation (location of gene is altered within the genome

• these structural change of a chromosome result in reshuffling of alleles on chromosome

Change in chromosome structure - Deletion

1. Deletion

• when a chromosome breaks in one or more places, and a portion of it is lost, the missing piece is referred to as deletion

• deletion can occur near one end or from the interior of the chromosome —> terminal or intercalary deletions

• effect of a deletion is profound, in which the genotype is altered due to the absence of certain gene loci

• if a deletion affects the same gene loci on both homologous chromosomes, the effect is usually lethal

• if only one of a homologous pair of chromosomes is affected, the effect on the phenotype is that the alleles on the non-deficient homologous will be expressed, even if recessive

Change in chromosome structure - Duplication

2. Duplication

• when any part of the genetic material is present more than once in the genome —> duplication

• can arise as a result of unequal crossing over between synapses chromosomes during meiosis or through a replication error prior to meiosis

• in the former case, both duplication and deletion are produced

• effects of duplications - gene redundancy, phenotypic variations

• in addition, duplications have also been deemed as an important source of genetic variation during evolution

Change in chromosome structure - Inversion

3. Inversion

• a segment of a chromosome is turned around 180º within a chromosome

• an inversion does not involve a loss of genetic information, but simply rearranges the linear sequence

• requires 2 breaks along the length of the chromosome and subsequent reinsertion of the inverted segment

• by forming a chromosomal loop prior to breakage, the newly created “sticky” ends are brought close together and rejoined

• organisms heterozygous for inversions may produce aberrant gametes that have a major impact on their offspring

• in addition, inversion may also result in position effects which lead to altered gene expression due to new positioning of a gene within the genome and thus play an important role in the evolutionary process

Change in chromosome structure - Translocation

4. Translocation

• movement of a chromosomal segment to a new location in the genome

• reciprocal translocation involves the exchange of segments between 2 non-homologous chromosomes

• genetic consequences of a reciprocal translocation are similar to inversions

• genetic information is not lost or gained —> rearrangement of genetic material

• may produce position effect (may realign certain genes in relation to other genes)

Change in chromosome number

• addition or loss of one or more chromosomes, addition of one or more haploid sets of chromosomes

Aneuploidy

• Aneuploidy: general condition in which an organism loses or gains one or more chromosomes, but not a complete set

• these chromosomal variations originate as a random error during the production of gametes

• non-disjunction is the failure of chromosomes or chromatids to disjoin and move to opposite poles during division

  • when this occurs in meiosis, the normal distribution of chromosomes into gametes is disrupted

• for the affected chromosome, abnormal gametes can be formed containing either two members or none at all

• the loss of a single chromosome from an otherwise diploid genome —> monosomy

• gain of one chromosome —> trisomy

• fertilising these with a normal haploid gamete produces a zygote with either three members (trisomy) or only one member (monosomy) of this chromosome

• non-disjunction leads to a variety of autosomal aneuploidy conditions in humans and other organisms

Monosomy

• monosomy in humans only occur for the X chromosome

• e.g. 45, X Turner syndrome - affected individuals have only 45 chromosomes, including just a single X chromosome

• 45, X (only 1 X copy present in the sex chromosome)

  • these individuals generally have female external genitalia and internal ducts, but the ovaries are rudimentary (underdeveloped)

  • short stature, skin flaps on the back of the neck, underdeveloped breasts, broad shieldlike chest

  • intelligence normal

• monosomy for any of the autosomes is usually not tolerated in humans or other animals

Trisomy

• e.g. 47, XXY Klinefelter syndrome - affected individuals have more than one X chromosome

• characteristics

  • genitalia and internal ducts that are usually male

  • but their testes are rudimentary and fail to produce sperm

  • generally tall and have long arms and legs and large hands and feet

  • although masculine developed does occur, feminine sexual development is not entirely suppressed

  • slight enlargement of breasts is common, hips are often rounded

• intersexuality, which may lead to abnormal social development

• intelligence often below the normal range

• e.g. Down syndrome - the only human autosomal trisomy in which a significant number of individuals survive longer than a year past birth

  • affected individuals have an extra chromosome 21

  • characteristics

    • short

    • skin folds over the corner of their eyes

    • stocky bodies

    • thick necks

    • prone to heart abnormalities

    • short life expectancy

    • low intelligence level

Polyploidy

• Euploidy - instances where multiples of the haploid chromosome set are found

• Polyploidy - instances where more than 2 multiples of the haploid chromosome set are found

• triploid - 3n chromosomes

• tetraploid - 4n chromosomes

• pentaploid - 5n

• well known in lizards, amphibians and fish

• more common in plants

Can originate in 2 ways

1. addition of one or more extra sets of chromosomes, identical to the normal haploid component of the same species resulting in autopolyploidy

2. combination of chromosome sets from different species may occur as a consequence of interspecific matings resulting in allopolyploidy

Autopolyploidy

• autotriploids occur in a few ways

• failure of all chromosomes to segregate during meiosis divisions (first-division or second-division non-disjunction) can produce a diploid gamete

  • if such a gamete survives and is fertilised by a haploid gamete —> zygote with 3 sets of chromosomes (3n)

• 2 sperms may fertilise an ovum —> triploid zygote

• produced under experimental conditions by crossing with tetrapolyploids

  • diploid organisms produce gametes with n chromosomes

  • tetraploids produce 2n gametes

  • upon fertilisation, the desired triploid is produced

• economically important triploid plants include several potato species of the genus Solanum, Winesap apples, commercial bananas, seedless watermelons, and the cultivated tiger lily Lilium tigrinum, which are propagated asexually

• autotetrapolyploids are theoretically more likely to be found in nature than autotriploids due to their even number of chromosomes

  • more likely to be found in nature than autotriploids due to

    their even number of chromosomes. Unlike triploids, which often produce genetically unbalanced

    gametes with odd number of chromosomes, tetraploids are more likely to produce balanced

    gametes when involved in sexual reproduction. Tetraploid alfalfa, coffee, peanuts and McIntosh

    apples are also of economic value due to either their larger sizes or more vigorous growth than

    their diploid or triploid counterparts.

Allopolyploidy

• results from hybridisation of 2 closely related species

• if a haploid ovum from a species with chromosome sets AA is fertilised by a haploid sperm from a species sets BB —> resulting hybrid is AB

A = a1, a2, … an

B = b1, b2, … bn

• hybrid plant may be sterile because of its inability to produce viable gametes

  • this occurs when some or all of the a and b chromosomes are not homologous and thus are unable to synapse in meiosis

  • leads to unbalanced genetic conditions

  • however, if the new AB genetic combination undergoes a natural or induced chromosomal doubling, 2 copies of all a chromosomes and 2 copes of all b chromosomes present —> pair during meiosis —> fertile AABB tetraploid produced

  • since this tetraploid contains equivalent of 4 haploid gametes derived from two separate species = allotetraploid

• when both species are known - amphidiploid (equivalent term)

Genetic screening

• Genetic screening is the analysis of a person’s DNA to check for presence of one or more alleles that are associated with disease

• samples of DNA can be obtained for testing from embryos, newborn babies, children and adults

• some screenings can be carried out before a baby is born

Genetic screening and genetic testing

• genetic test - link to family history

  • e.g. If a person from a family with Huntington’s disease, for example, begins to develop symptoms of

    the disease, he or she is likely to be advised to have a genetic test (KIV: Molecular Techniques) to

    confirm the diagnosis. Other people in the family who do not show any symptoms may also be

    offered the opportunity to have the genetic test

  • is the same whether a person has symptoms or not - involves PCR to amplify the gene HTT in the case of Huntington’s diseases and electrophoresis to determine the lengths of the two alleles and to count the number of CAG repeats

• genetic screening - people who are free of the symptoms of a disease

Maternal genetic screening

• maternal genetic screening, also known as prenatal genetic screening, are tests that provide information about the health of a foetus

• check for congenital conditions e.g. Down Syndrome, trisomy-13 and spinal bifida

• high-risk pregnancies

  • over age of 35

  • family or personal history of genetic conditions

  • history of miscarriage or stillbirth

  • lifestyle factors e.g. exposure to cigarette smoke, radiation

• results of screening tests can be used for counselling purposes to help parents make informed decisions about the outcome of pregnancy

• maternal genetic screening tests do not diagnose medial conditions

• these screening tests determine if the foetus is at an increased or decreased risk of a particular abnormality

Prenatal Diagnostic tests

• diagnostic test is only performed when a screening test is abnormal

• prenatal diagnostic tests are usually more invasive tests that may pose a higher risk to the pregnant woman and the foetus

Maternal genetic screening for down syndrome

Down syndrome (trisomy-21)

• non-disjunction is found in 95% of down syndrome, the other 5% caused by translocation, mosaicism or partial trisomy

• occurs when there is additional chromosome for the 21st pair of chromosomes

• maternal genetic screening is used to look for potentially at-risk pregnancies

• maternal genetic screening has significantly reduced the rate of down syndrome live briths over the years in singapore

Bioethical considerations in maternal genetic screening