Week 6 Flashcards
Types of variation in genome
-alterations in the sequence of bases in a specific section of DNA: single nucleotide polymorphisms (change in single base), small deletions or duplications (few bases)
-microsatellites (tandem repeats of 2-6 bp)- <100bp in total length
- minisatellites (variable number tandem repeats of 10-60bp)- can span several kb
-Larger deletions/duplications (copy number variation) of DNA segment, or segments of chromosomes
-Changes in number or structure of chromosomes
Variation in genome can lead to
Altered effects of a protein or control of genes leads to:
Normal human variation, differences in response to medication, influence the likelihood of disease, directly result in a genetic condition
How variation in genome affects health and disease depends on its type and where it is
How can a genome variant be classified
Size
Frequency -common or rare
Clinical effects- non-pathogenic (no change in phenotype) or pathogenic (disrupt gene function, clinical effect)
What is a mutation
An alteration or change in the genetic material
From exposure to mutagenic agents but more arise spontaneously through errors in DNA replication/repair
More likely to be recognised if effects are detrimental some are not recognised
DNA sequence variants
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
What is a SNP
A change in a single base at a particular position
To be called an SNP, a base change has to have frequency of >1%
How can the genome be examined
Bases (small region)— sequencing or microarray analysis
Large blocks DNA— microarray analysis, fluorescence in situ hybridisation FISH
Chromosomal— light microscopy
DNA sequencing
Developed by Fred Sanger 1977
Use technology to read order of bases and compare to reference sequence
Amplify small amounts of target DNA usually by PCR
DNA is used as a template to generate a set of fragments that differ in length form each other by a single base
The fragments are then separated by size and the bases at end are identified, recreating original sequence of DNA
Why sequence DNA
Sequencing DNA determines exact position of mutation within gene
Determines the type of mutation (including single base changes)
Nucleic acid sequencing by chain termination is now automated
The use of fluorescently-labelelled ddNTP terminators has allowed automation and high-throughput sequencing
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
Next generation sequencing
Whole molecule sequencing in one reaction
Much faster and cheaper than Sanger sequencing
Patients can have genome sequenced- make predictions about health, make diagnosis, find out which medications they’re most likely to respond to
When do mutations occur
Cell division
From intrinsic and extrinsic attacks on DNA
How can errors in DNA replication and Meiotic division cause human disease
Mutation is in gametes so is passed onto offspring
How can errors in DNA replication and mitotic division cause human disease
Passed onto daughter cells
Mitosis is used for growth of embryo and for maintenance of tissues. Somatic changes
In body cells
Endogenous mechanisms causing DNA damage
Depurination: spontaneous fission 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 A in new strand
Reactive oxygen: attack purine/pyrimidine rings
Methylation of cytosines: at CpG dinucleotides spontaneous deamination of 5-methyl-cytosine to thymine
A common mechanism of mutation: C to T at CpG
High frequency of C to T transitions in genome
Especially at CpG dinucleotides- can become methylated so C more likely to be spontaneously deaminated to a T
CpG to TpG mutations
Cytosines at CpG sequences are frequently methylated; 5-methyl-cytosine deaminated to thymine
Mutation rate at CpG 8.5x more likely than that of other dinucleotides
Frequent effect is production of a nonsense mutation: CGA—TGA arginine to stop codon
Extracellular agents causing DNA damage
-ultraviolet light
-environmental chemicals (interpolate into DNA or cause DNA breaks or chromosome aneuploidy)
-ionising radiation (causes breaks in DNA)
Ultraviolet light on dna damage
In presence of uv light two adjacent thymine bases covalently attach to each other forming a thymine dimer
Thymine dimers disrupt 3D structure and can stall DNA replication machinery
Bases damaged by sunlight are excised and the strand resynthesised
Where a mutation occurs has potential implications for offspring
Germline mutations- mutation in egg or sperm, meiosis, all cells affected in offspring, heritable
Somatic mutations- occur in non-germline tissues, specific tissues, mitosis, non heritable
Why are there checkpoints in cell division
Attempt to prevent mutations being passed on to daughter cells
G1- is environment favourable?
G2- is all DNA replicated?, is all DNA damage repaired?
Checkpoint in mitosis- are all chromosomes properly attached to mitotic spindle?
Correcting DNA replication errors
DNA replication machinery has proof reading
DNA polymerase adds a base, checks it, excises it if wrong, moves on
DNA mismatch repair system corrects 99% of residual errors from replication machinery
Replication copy errors leave mispaired nucleotides would cause permanent mutation when strand with error is copied in next round
Protein complex recognises DNA mismatch, excises newly synthesised mismatched strand and uses original template strand to re-synthesis new strand
Mismatch repair system mutations
As mismatch repair system is protein complex it’s coded for by genes
Mutations in mismatch repair genes themselves lead to accumulation of somatic mutations and so predispose to cancers
Damage to DNA due to ionising radiation and reactive oxygen species
Harmful, causes double stranded breaks- dont have a template strand
Repaired by either- the sequence from other homologous chromosome of pair used to synthesise missing DNA, accurate method (homologous recombination) or by end joining broken ends (Error prone), leads to deletion of nucleotides at repair site
Broken ends processed by nuclease, end joining by DNA ligation
Types of mutations affecting coding sequence
Missense
Nonsense
Frameshift
Duplication
Deletion
Insertion
Varying effects on health depending on where they occur and whether they alter the function of essential proteins
Pathological mutations associated with protein coding genes
Exons
Intragenic non-coding sequences necessary for correct gene expression
Can get variation in intronic regions but less likely to be harmful but can be pathogenic if in regulatory sequences
Silent mutations/ sequence variant
Single base substitutions
A base pair change that does not change the amino acid sequence, no effect
Can create a cryptic splice site
Missense mutation
Single base substitution
Changes to a codon for another amino acid can be harmful mutation or polymorphism with a neutral effect
Amino acids differ in size, polarity , side chains
The more similar the amino acid is the less effect on protein structure, less serious
Nonsense mutation
Single base substitution
Change from an amino acid codon to a stop codon
Shorter protein- can’t act in same way
Pathogenic
Aberrant transcript usually degraded by ‘nonsense mediated decay’ process, end up without any protein
Splice site mutations
Single base substitutions
Takes place where splicing occurs, altered mRNA
A change that results in altered RNA sequence
Mutations at splice sites can cause exon skipping or incorporation of intron sequence into mRNA
Mutation at acceptor splice site or splice donor site
At acceptor site deletes exon from mRNA, no longer recognised as an acceptor site so skips to next one deleting exon in middle
Can change size and content of protein
Frameshift mutations
Insertion or deletion of base pairs, producing stop codon downstream
Affects sequence after mutation, DNA sequence shifted
Mainly pathogenic
A shortened altered protein may be expressed or mRNA degraded by nonsense mediated decay system
Copy number variants
Small arrays of triplet repeats in coding sequences of genes are prone to expand in number and disrupt function of gene
Short tandem repeats can mispair and cause pathogenic deletions and insertions which cause Frameshift
Expansion of number of STRs within or in vicinity of a gene can affect gene expression
Repeats can predispose to large deletions and duplications
Larger deletions and insertions
Usually caused by unequal crossing over between repeat sequences, caused by misalignment
May affect a genes, several genes or section of chromosome- vary in size
Clinical effects depend on genes involved and gene dosage
Deletion of a segment tends to be more severe on phenotype
What is the total human genome made up of
DNA sequences on one chromosome from each of the 22 autosome pairs, both sex chromosomes and the mitochondrial genome
Chromosomes, genes and dna
A chromosome is made of a single molecule of DNA
A specific stretch of DNA where the sequence contains genetic instructions is a gene
Genes are arranged one after another along DNA of chromosome with stretches of non coding DNA between them
Genes are arranged in linear order on chromosomes
Chromosome structure as seen at metaphase
Telomere- DNA and protein cap, ensures replication to tip, tether to nuclear membrane
Short arm (p) and long arm (q), long arm is after centromere
Centromere- joins sister chromatids, essential for chromosome segregation at cell division
Light bands- replicate early in S phase, less condensed chromatin, transcriptionally active, gene and GC rich
Dark (G) bands- replicate late, contain condensed chromatin, AT rich
Light and dark bands alternate
How do we examine chromosomes
Karyotype- low resolution
Fluorescent in situ hybridisation FISH
Array CGH -highest resolution
How do we obtain a karyotype from peripheral lymphocytes
Take blood sample, separate off red blood cells, add culture medium to white cell suspension, incubate 3 days at 37, colchicine added (stop each cell dividing), separate off white cells, hypotonic saline added, cells fixed, cells spread onto slide by dropping, stained, photographed, karyotype
Analysing karyotype
Recognise each chromosome by their individual binding pattern
Ensure there is the right number of chromosomes
Look for large copy number variants, any duplications, deletions
Change in structure or location of genetic material
FISH
Allows you to look at segments of chromosome at higher resolution
In karyotype you can only detect large deletions etc
Fluorescent probes that hybridise to specific area of chromosomes
Can give different chromosomes different colour fluorescent probes, look at number of each
Used when you know what you’re looking for
Array CGH-comparative genomic hybridisation
Most common
High resolution
Looks at all genetic material not just targeted
Takes advantage of fact DNA hybridises
Use test genomic DNA (patient DNA) and cohybridise with normal reference DNA, fluorescent dye them different colours e.g green and red
DNA microarray containing probes representing genomic regions of interest, DNAs compete for same probe sites on microarray, assess colour ratios, look for deletions & duplications in chromosomes much smaller that what we would’ve previously seen
What types of chromosome abnormalities are there
Abnormalities of chromosome number
Abnormalities of chromosome structure
Chromosome abnormalities are also classified according to which cells of the body they’re distributed in:
Constitutional= all cells of body
Somatic= only in certain cells/ tissues of body
The karyotype international description
- Total number of chromosomes
- Sex chromosome constitution
- Abnormalities/ variants
E.g. 47,XX,+21= trisomy 21
Abnormalities of chromosome number
Aneuploidy- changes in single chromosome number
Trisomy- additional chromosome
Monosomy- missing chromosome
Polyploidy- change in overall chromosome numbers
How can abnormalities arise in cell division- meiosis
Nondisjunction during meiosis II or I
Paired chromosomes don’t separate leading to trisomy
Disomic and nullisomic gametes
Parental origin of Meiotic error leading to trisomies
Nondisjunction events increase as women get older
The stock of oocytes is ready by 5 months gestation, each remains in maturation arrest at crossing over stage until ovulation, don’t complete meiosis until woman goes through puberty. Lengthy interval between onset and completion of meiosis
Older a women gets the older the eggs
Accumulating effects on primary oocyte during this phase may damage cells spindle formulation and repair mechanisms predisposing to Nondisjunction
Clinical consequences of abnormal chromosome number
Significant development abnormalities occur due to the imbalance of gene products
The effects of reducing the gene copy usually has more severe consequences than increasing gene copy
Only types of abnormalities of chromosome number that survive are those affecting sex chromosomes or trisomy 21, 18, 13
Trisomy or monosomy of other autosomes will not survive due to large gene content
Most frequent numerical anomalies in liveborn
Autosomes: Down syndrome (trisomy 21), Edwards syndrome (trisomy 18), Patau syndrome (trisomy 13)
Sex chromsomes: Turner syndrome (X), Klinefelter syndrome (XXY)
All chromosomes: Triploidy (69 chromosomes)
The genetic basis of Down syndrome
95% have trisomy 21
4% have an extra copy because of Robertsonian translocation
1% have mosaicism with normal and trisomy 21 cell lines
How does having 3 copies of genes on chromosome 21 cause features of Down syndrome
Gene dosage effect- features of syndrome caused by 1.5x amount of specific gene products from chromosome 21
Amplified developmental instability- features caused by overall effect of imbalance on development
Implications for genetic counselling
Refers to the process of giving people information about genetic risk in their family so they can make informed decisions
Aneuploidy is usually results of Meiotic non disjunction related to maternal age, the risk of recurrence is therefore low and will be related to age of mother. Not normally family history, not inherited
Abnormalities of chromosome structure
Translocations: reciprocal/ Robertsonian
Deletion
Duplication
Inversions
Ring chromosome
Marker chromosome
Complex rearrangements
Robertsonian translocations
Breakage of two acrocentric chromosomes (13,14,15,21,22) at or close to their centromeres, with subsequent fusion of their long arms-short arms are lost
What are acrocentric chromosomes
Chromosomes where the centromere is near top of chromosome, have a very small short arm
Balanced Robertsonian translocation
All chromsomes are there but in different position, Same number of each
Doesn’t affect phenotype as there is no gene imbalance
Occurs in 1/1000
Robertsonian translocation unbalanced
Can lead to Down syndrome and patau syndrome
Different number of chromosomes
Gene imbalance so affect on phenotype
Acrocentric chromsomes are a different size and shape to others
What happens when a balanced carrier has children
The two normal chromosomes and the Robertsonian translocation chromosome can segregate in 3 ways
Normal, balanced carrier (no change in phenotypes)
Trisomy, monosomy etc- genetic imbalance is so large baby wouldn’t survive- miscarriages or person can’t get pregnant
Translocation Down syndrome
Translocation trisomy 14, monosomy 14, monosomy 21 are lethal
Reciprocal translocation
2 non paired chromosomes (non homologous) break and exchange fragments
Named based on which centromere they contain
E.g. der(3)
Any chromosomes can be involved not just 13,14,15,21,22
Balanced reciprocal chromosome translocation
No gain or loss of genetic material, just changed position, carrier
Between two non homologous chromosomes
Moving position
If you’re a carrier there’s a risk of having a child with unbalanced gamete as chromosomes can segregate in different ways during meiosis
Normal, balanced carrier (no change to phenotype)
Partial trisomy+partial monosomy of each chromosome which can lead to miscarriage if imbalance is large, or congenital malformation, developmental delay, mental abnormality
The size and position of chromosome segments involved in translocation may have an effect on
The pairing of chromosomes at meiosis
Frequency of different forms of the translocation in gametes
The likelihood of the conceptus with that abnormality developing to term
These depend on the effects of the genes on translocated segments and amount of chromosome imbalance
Large chromosome deletions/duplications
Occur because of unequal crossing over in meiosis following mispairing often at sites of repeated sequences
Effects vary depending on size of imbalance
Mosaicism
Two populations of cells with different genetic constitutions usually as a result of an error in mitosis
Occurs because of a genetic change that occurs after cell division, occurs postzygotically
Mutation in single gene, chromosomal anomaly
in mitotic cell division so all daughter cells have genetic change
Somatic mosaicism
Two populations of cells in body
Gonadal mosaicism
Two populations of cells only in gonads
