mutations + genetics Flashcards
nucleolus
Site of transcription and assembly of rRNA
chromatin
mixture of DNA, proteins and RNA that package DNA within the nucleus.
It is divided between 2 forms
heterochromatin (condensed) - transcriptionally inactive
euchromatin (extended) - transcriptionally active
mechanisms by which chromatin is made more accessible
Histones can be
- enzymatically modified
- displaced by chromatin remodeling complexes
- These processes are reversible
DNA Replication
semi-conservative
i.e. one-half is old; one-half is new.
bi-directional
i.e. replication in two directions
(leading & lagging strand)
DNA replication steps
(usage of DNA polymerase)
[1] Proteins unzip the double helix by breaking the hydrogen bonds
[2] New nucleotide molecules are then paired with the two DNA strands.
Leading strand:
Synthesizes DNA in a 5’ to 3’ brine direction in one go
Lagging strand:
Synthesizes DNA in 5’ to 3’ brine direction using a series of short fragments [Okazaki fragments]
Stiched together by DNA ligase once completed
Uses RNA primer in between the series
alternative splicing
exons of the RNA produced by transcription of a gene (a primary gene transcript or pre-mRNA) are reconnected.
The resulting different mRNAs may be translated into different protein isoforms;
thus, a single gene may code for multiple proteins.
tRNA and rRNA
tRNA - acts as an adapter molecule between the coded amino acid and the mRNA.
-role is to translate mRNA sequence into amino acid sequence.
rRNA - combines with proteins to form a ribosome
- Produced in the nucleus & transport to cytoplasm
sickle cell anaemia
an inherited blood disorder
a single nucleotide substitution leads to a pathological phenotype by changing the expressed amino acid thus changing the structure of the protein.
Adenine to Thymine substitution leads to → encoding changes from Hydrophilic Glutamate to Hydrophobic Valine
chromosome abnormalities
Numerical
→ wrong number of chromosomes in a karyotype
Structural
→ large scale rearrangement of chromosomes in a karyotype
Mutational
→ small scale changes from deletions to base pair changes
mutation terminology
Monosomy - the state of having a single copy of a chromosome pair instead of the usual two copies
Trisomy - a chromosomal condition characterised by an additional chromosome.
→ 47 chromosomes instead of 46
→ related to first trimester miscarriages
Triploidy - a condition in which there are three complete sets of chromosomes in a single cell
→ 69 chromosomes
Tetraploidy - a condition in which there are four complete sets of chromosomes in a single cell.
→ 92 chromosomes
Why does down syndrome incidence go up with increasing maternal age?
Because of increases in nondisjunction during meiosis
→ egg cells sitting in meiosis I for decades…
nondisjunction
failure of homologous chromosomes or sister chromatids to separate properly during cell division.
autosomal and sex chromosome aneuploidy syndromes
autosomal
down syndrome
patau syndrome
edwards syndrome
sex chromosome
turner syndrome
klinefelter syndrome
downs syndrome
Trisomy 21
– Incidence: 1 in 700
* Increases with advancing maternal age
– Characteristic facial dysmorphologies
– IQ less than 50
– Average life expectancy (50-60 years)
– Alzheimer’s disease in later life
– Chromosomal findings
* Trisomy 21: non-disjunction (95%), usually maternal origin
* Unbalanced Robertsonian translocation (4%)
* Mosaicism (1%)
patau syndrome
Trisomy 13
– Incidence: 1 in 5000
– Multiple dysmorphic features and mental retardation
– About 5% die within first month, very few survive beyond first year
– Non-dysjunction (90%), maternal origin
– Unbalanced Robertsonian translocation (10%)
edwards syndrome
Trisomy 13
– Incidence: 1 in 5000
– Multiple dysmorphic features and mental retardation
– About 5% die within first month, very few survive beyond first year
– Non-dysjunction (90%), maternal origin
– Unbalanced Robertsonian translocation (10%)
turner syndrome
45,X
– Incidence: 1 in 5000 to 1 in 10000 (liveborn)
– Incidence at conception much greater, about 97% result in spontaneous loss
– Females of short stature and infertile
– Neck webbing and widely spaced nipples
– Intelligence and lifespan is normal
klinefelter syndrome
47,XXY
– Incidence: 1 in 1000
– Tall stature, long limbs
– Male but infertile, small testes, about 50% gynaecomastia
– Mild learning difficulties
structural abnormalities
characterized by gross large scale rearrangement in the karyotype.
Balanced or unbalanced rearrangements
Can occur through:
Translocations
– Reciprocal: involving breaks in two chromosomes with formation of two new derivative chromosomes
– Robertsonian: fusion of two acrocentric chromosomes
Deletions - a part of a chromosome is left out during DNA replication
Insertions
Inversions - a chromosome rearrangement occurs in which a segment of a chromosome is reversed end to end.
balance translocation
when the same amount and number of copies of the original DNA are still present after a part of a chromosome has broken off and reattached in another location.
i.e. there is no DNA missing or additional DNA
unbalanced translocation
when a different amount and number of copies of the original DNA are present after a part of a chromosome has broken off and reattached in another location.
i.e. there is DNA missing or additional DNA
genetic mutation
Germline or
gene disruption
Somatic
disease-associated
Polymorphism
– No phenotypic effect
– Frequency >1%
types of mutations
[A] Non-coding
→ could affect promoters or regulatory molecules
[B] Coding:
- Silent
– e.g. CGA (Arg) to CGC (Arg)
[2] Missense
– e.g. CGA (Arg) to GGA (Gly)
[3] Nonsense
– e.g. CGA (Arg) to TGA (Stop)
[4] Frameshift (deletion or insertion)
– e.g. CGA (Arg) to CCGA (Pro, then out-of-frame)
mutation detection
- Polymerase chain reaction (PCR)
- Gel electrophoresis
- Restriction fragment length polymorphism (RFLP) analysis
- Amplification refractory mutation system (ARMS)
- DNA sequencing
PCR
aim is to amplify a specific piece of double stranded DNA for analysis
What do we need for PCR?
- Sequence information
- Oligonucleotide primers
- DNA
- Nucleotides
- DNA polymerase
PCR technique
[1] Denature
→ when the double-stranded template DNA is heated to separate it into two single strands.
[2] Anneal
→ when the temperature is lowered to enable the DNA primers to attach to the template DNA.
[3] Extend
→ when the temperature is raised and the new strand of DNA is made
gel electrophoresis
used to separate DNA fragments by size.
- Apply an electric field
- DNA is negatively charged
- Separate through agarose gel matrix
- Visualise DNA fragments
PCR Gel advantages
- Speed
- Ease of use
- Sensitive
- Robust
PCR applications
- DNA cloning
- DNA sequencing
- In vitro mutagenesis
- Gene identification
- Gene expression studies
- Forensic medicine
- Typing genetic markers
- Detection of mutations
Amplification Refractory Mutation System (ARMS) analysis
only normal alleles
→ amplification with normal primers
normal & mutant alleles (heterozygote)
→ amplification with both normal & mutant primer
only mutant alleles
→ amplification with mutant primers
advantages/ disadvantages of Amplification Refractory Mutation System (ARMS) analysis
cheap
easy to use
specific and sensitive
detects certain mutations
unreliable
Restriction fragment length polymorphism (RFLP) analysis
→ Test whether samples contain mutant or normal allele
- Restriction enzyme that recognizes & cuts normal DNA but not mutant DNA
Restriction endonucleases
Enzymes from bacterial cells that degrade DNA of invading viruses
- Protective mechanism
- Recognise specific DNA sequences
- Usually 4-8 bp
- Always cut DNA at the same site
advantages and disadvantages of RFLP
- Simple
- Cheap
- Non-radioactive
- Requires gel electrophoresis
- Not always feasible
DNA sequencing gold standard
Chain termination method (Sanger sequencing)
Use of dideoxynucleotides
- chain-elongating inhibitors of DNA polymerase
→ causes chain termination & stops DNA polymerase
Advantages and disadvantages of Sanger method for DNA sequencing:
Gold standard for mutation detection
Automation and high throughput
Expensive equipment
Poor quality sequence read
– First part of sequence (15 to 40 bases)
– Deterioration after 700-900 bases
fitness
the relative ability of organisms to survive (long enough) to pass on their genes.
Factors that influence it:
[1] Alleles
- not at all in most cases (neutral allele)
- sometimes decrease (deleterious allele)
- rarely increase (advantageous allele)
[2] Child background
- Role of grandparents / family / clan / society in food sourcing,
- Defence against invaders, keeping child safe from harm and education
mutations in recessive genes
rarely affects carriers
common recessive diseases
* Sickle cell disease
* Thalassaemia
– If it does, occurs with a selective pressure
* e.g. malaria resistance (SCD carriers)
* e.g. plague or cholera resistance (cystic fibrosis)
– De novo recessive mutation uncommon as a cause of disease
– Mutation in dominant and X-linked genes can be inherited or de novo
de novo mutation
A genetic alteration that is present for the first time in one family member
common in dominant disorders
esp. where disease reduces reproductive fitness
e.g. common cause of severe learning disability
- Up to 1/3 of lethal X-L cases are due to a de novo mutation
Hardy-Weinberg equilibrium (HWE)
Allele frequencies remain constant generation to generation.
Relative proportion of genotype frequencies remain constant generation to generation
Assumptions underlying HWE:
- Mutation can be ignored
- Migration is negligible (No gene flow)
- Mating is random
- No selective pressure
- Population size is large
- Allele frequencies are equal in the sexes
mutation and migration (gene flow)
→ Mutations increase the proportion of new alleles.
→ Introduction of new alleles as a result of migration or intermarriage leads to new gene frequency in hybrid population.
non random mating
Non-random mating - leads to increased mutant alleles, thereby increasing proportion of affected homozygotes.
[1] Assortative mating
- Choosing of partners due to shared characteristics
Deafness & sign language
[2] Consanguinity
- Marriage between close blood relatives.
Cultural pressures for inter-marriage within clans / religions etc.
founder effect
new migration from physical or cultural isolation
leads to a change in the frequency of an allele within a population.
It occurs when a small group of migrants that are not genetically representative
of the population from which they came, establish in a new area.
Leads to reduction in genetic variation
e.g. group of 12 migrate to an area and half are sickle cell carriers
eventually due to isolation it would become more common
so = less genetic variation
natural selection
a process by which biological traits become either more or less common in a population.
[1] Negative selection
- Reduces reproductive fitness.
- Decreases the prevalence of traits.
- Gradual reduction of mutant allele.
[2] Positive selection
- Increases reproductive fitness.
- Increases the prevalence of adaptive traits.
- Heterozygote advantage.
genetic drift
- random fluctuation of one allele transmitted
to high proportion of offspring by chance.
causes founder effect
bottleneck effect
a sharp reduction in the size of a population due to environmental events
e.g. famines, earthquakes, floods, fires, disease, and droughts or human activities such as genocide and human population planning.
cancer from gene mutations
Somatic mutations
* Occur in nongermline tissues
* Are nonheritable
Germline mutations
* Present in egg or sperm
* Are heritable
* Cause cancer family syndromes
protoncogenes
normal gene that codes for proteins to regulate cell growth and differentiation.
- Mutations can change it into an oncogene
- Oncogenes can accelerate cell division
- Cancer arises when cell is stuck in “on” mode
oncogenes
dominant genes in effect, they are derived from a mutation in proto-oncogenes.
→ accelerate cell division
→ one mutation sufficient for role in cancer development
tumour suppressor genes
recessive genes in effect that inhibit cell cycle or promote apoptosis or both
→ The cell’s brakes for cell growth
→ Cancer arises when both brakes fail
DNA damage-response genes
genes responsible for repair mechanisms for DNA
- Cancer arises when both genes fail, speeding the accumulation of mutations in other critical genes
mismatch repair genes
genes that correct errors that spontaneously occur during DNA replication
like single base mismatches or short insertions and deletions
If they are functioning abnormally → accumulation of errors → microsatellite instability
microsatellites
(aka Simple Sequence Repeats SSR) are repeated sequences of DNA,
can be made of repeating units of 1 – 6 base pairs
Hereditary nonpolyposis colorectal cancer (HNPCC) results from failure of MisMatch Repair genes (MMR)
clinicians look/ screen for hnpcc
To prevent it from developing into a metastatic cancer by removing it in the Adenoma stage.
cancer types
[1] BENIGN – lacks ability to metastasize.
Rarely or never become cancerous.
Can still cause negative health effects due to pressure on other organs.
[2] DYSPLASTIC – ‘benign’ but could progress to malignancy.
Cells show abnormalities of appearance & cell maturation.
Sometimes referred to as ‘pre-malignant’.
(NB distinguish from ‘hip dysplasia’ which is macroscopically abnormal but not pre-malignant!)
[3] MALIGNANT – not ‘benign’. Able to metastasize.
(distinguish from ‘malignant hypertension’, ‘malignant hyperthermia’)
