Mutations In Repetitive Dna

Front 1

What are three types of mutations triggered by repetitive DNA?

Back 1

Copy number variation

PathogenicMinisatellites

Pathogenic Microsatellites (Dynamic mutations)

Front 2

Overview of CNV

What is it?

Inheritance

Cause

Analysis

When is it disease causing

Back 2

Variable gene/chromosomal fragments

Inherited or de novo

LCR

Microarrays (CGH), also FISH, karyotype

Pathogenic when gene dosage, coding or regulatory sequence.

Recombination, replication and retro-transposition

Front 3

What are CNV?

Back 3

· Deletions or duplications in the genome – in a gene,pseudogene, chromosomal fragment, non-coding DNA

Front 4

What % of all DNA is copy number variable?

Back 4

12%

Front 5

What is the most common origin of CNVs?

What are complications

Back 5

· Largely inherited (meiotically),

Non-allelic homologous recombination events in which unmatched regions of chromosomes are mistakenly recombined during meiosis

· However, studies have revealed extensive CNV between different cells in the same individuals - must have arisen post-fertilisation

Also, some complex genetic rearrangements (discontinuous duplications mixed with deletions, inverted duplications, and triplications) cannot be readily reconciled with a non-allelic homologous recombination mechanism-

microhomology-mediated break-induced replication (MMBIR) model

Front 6

Explain de novo CNVs

Back 6

Can be de novo (somatically) – identical twins can differ in CNV.

Arise through diverse mechanisms at various stages of development

Front 7

Which segments of DNA are most susceptible to CNV?

Back 7

Junctions of recurrent CNVs are found in LCR, Region-specific repeat sequences. More susceptibility to genomic rearrangements. Results in CNVs. Size, orientation, % similarity and distance between copies influence susceptibility of LCRs to genomic rearrangement.

Segmental Duplications (SDs) map near ancestral duplication sites in a phenomenon called duplication shadowing which describes the observation of a ~10 fold increased probability of duplication in regions flanking duplications versus other random regions

CNV in microsatellites can arise through replication slippage and defective mismatch repair

Front 8

What are LCR?

Back 8

· LCR (low copy repeats) = Segmental duplications –ancestral sequences which have been duplicated. Euchromatic regions close tocentromeres and telomeres are unstable and prone to recombination with otherchromosomes.

Front 9

What is the size of CNV?

What is the % sequence identity

Back 9

· CNV > 1kb-millions in length sharing >90% sequenceidentity

Front 10

When do CNV cause disease?

Back 10

· causing when a gene is dosage sensitive, coding sequence or regulatory sequences involved. Clustering in regions of complex patterns of direct and inverted LCR may relate to absence of dosage-sensitive genes

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What are mechanisms of CNV?

Back 11

Intrachromatid recombination between direct LCR repeats.

Recombination at LCR regions of misaligned sister chromatids or homologous chromosomes

Recombination at LCR regions of misaligned non-homologous chromosomes- reciprocaltranslocation.

Front 12

Explain MMBIR

Back 12

single-strand 3′ tails from broken replication forks will anneal with microhomology on any single-stranded DNA nearby, priming low-processivity polymerization with multiple template switches generating complex rearrangements, and eventual re-establishment of processive replication.

Front 13

Explain Intrachromatidrecombination between direct LCR repeats.

Back 13

Meiosis.

Mispairing between LCRs due to high sequence homology so crossover changes the orientation – inversion

Front 14

Explain Recombinationat LCR regions of misaligned sister chromatids or homologous chromosomes.

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Direct repeats – head of one repeat joins tailof its neighbour. Inverted repeats – joining of heads or tails.

Over longtime-scale, duplicated sequences can be separated on same chromosome (by DNA insertion/inversion)or distributed on different chromosomes by translocations.

Front 15

What is the simplest method to detect pathogenic CNVs?

Back 15

Karyotype

Front 16

Explain karyotype analysis

Back 16

· Detects trisomy 21. Arrests in metaphase during colchicine. Light bands – euchromatin, dark bands – heterochromatin; uniqueGiemsa pattern.

· Results: can detect aberrations as small as 5 Mbwith banding analysis.

Front 17

Explain Array-comparativegenomic hybridisation microarrays

Back 17

· Extract genomic DNA, process through restriction digestion and fragmentation. Perform competitive hybridization between 2 DNA samples (labelled with two difference fluorophores) onto nucleic acid targets. Targets can genomic fragments cloned by BACs or plasmids. BACs - most comprehensive coverage

Red fluorophore (cyanine 5), reference DNA green (cyanine 3).

Detect CNS by quantifying relative fluorescence intensities of each hybridised fluorophores. If equal intensities - same DNA.

Front 18

How are the results obtained forArray-comparativegenomic hybridisation microarrays?

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Use confocal laser. Yellow – no change in CNV. Red –high copy number in control; deletion in sample. Green – higher copy number in test; duplication in sample as no DNA binding to the probe

· Fluorescent signal intensity of labelled test DNA relative to reference DNA: linearly plotteda cross each chromosome – identify copy number changes

Front 19

Pros/Cons of Array-comparativegenomic hybridisation microarrays

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· Ads:Quickly scans entire genome. Does not require cells that are undergoing division

· Dis: Resolution limited to 5-10 Mb alterations by metaphase FISH

Front 20

FISH - when is it used?

Example

Back 20

Looking for specific CNV

William's syndrome -Deletion of elastin gene. Dual colour FISH using a specific and control probe. Only one copy of elastin gene (only on one chromosome 7). UV to visualise.

Front 21

Explain FISH

Back 21

· Probe is fluorescently labelled DNA fragment.
Denature DNA in situ.

Homogenous DNA probe


Heterogenous· Chromosomal painting – picks up large duplications and rearrangements. Complex mixture of many different types of DNA fragments from one type of chromosome

Front 22

What are pathogenic CNVs?

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· 50% of haemophilia A. Intrachromatid recombination between inverted LCR repeats – inversions.

· Paternal 15q12 deletion causes Prader Willisyndrome. Maternal 15q12 deletion causes Angelman syndrome

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What are features of Minisatellites?

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· Occur at more than 1,000 locations in human genome. High mutation rate and high diversity.

· Multi locus or single locus

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What are VNTR?

Back 24

Variable number tandem repeats

· Minisatellites and microsatellites

Front 25

What is the structure of minisatellites?

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· Repetitive DNA – certain DNA motifs (10-60 bps) are repeated 5-50 times. · Generally GC-rich, repetitive motifs

Front 26

What are hypervariable minisatellites?

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core units 9–64 bp long and are found mainly at the centromeric regions

1% prevalence

Front 27

Where are minisatelittes prominent?

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Centromeres and sub-telomeric region (90% in humans) of chromosomes. The telomere itself is a tandem repeat – TTAGGG – hexanucleotide repeat

Front 28

What is the function of minisatellites?

Back 28

· Regulators of gene expression – transcription,alternative splicing, imprint control

· Generally non-coding but sometimes are part of possible genes

Front 29

Give an example of pathogenic minisatellites

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Dodecamer repeat expansion at promoter region of CSTB, cystatin B gene - inhibits cathepsins

Causes inherited(autosomal recessive) progressive myoclonus epilepsy. Unaffected 2-3 copies,affected >40.

Front 30

What is the use of minisatellites?

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DNA fingerprinting - due to high level of polymorphism

Front 31

Explain mutability of minisatellites

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· Proposed that Minisatellites sequences encourage chromosomes to swap DNA.

· In alternative models, it is the presence of neighbouring double-strand hotspots which is the primary cause of minisatellite repeat copy number variations

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Where are microsatellites located?

Back 32

· Distributed through the genome.

Many located in introns – biologically silent. Accumulates mutations unhindered over generations.

· Others located in regulatory flanking or intronic regions of genes – lead to phenotypic changes, notably triplet expansion diseases. E.g. Fragile X – neurodegenerative disorder. IN the promoter of the gene

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Example of Pathogenic Microsatellites

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· Expansion of CAG triplet repeat stretch within Huntingdon’s gene results in a different form of protein – damages brain cells.CAG for glutamine – series is polyglutamine tract

the more repeats, the more severe the disease and the earlier its onset

Front 34

Explain CAG repeats

Recent results

What mode of inheritance

Back 34

Nine neurologic disorders - typically in coding regions of unrelated proteins

polyglutamine tract - subject to increased aggregation

Recent results suggest that the CAG repeats need not always be translated in order to cause toxicity.

autosomal-dominant

Front 35

Explain triplet repeat expansion disorders

Why three nts?

Back 35

insertion mutations. dynamic mutations

Three nt - tend to be near coding regions, other multiples cause frameshifts - developmental lethality.

Three nt - only cause frameshift if stop codon is triplet added (TGA, TAG, TAA).

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What is the structure of Pathogenic Microsatellites

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· DNA motifs (2-5 bps) are repeated, 5-50 times.

· Trint repeat disorders (GTCGTC…) show genetic anticipation – severity increases with each successive generation that inherits them due to addition of further CAG-repeats. Huntington’s – more than 36 CAG repeats

36–40Reduced Penetrance+/- Affected

>40Full PenetranceAffected

Front 37

What are two causes of mutations in pathogenic microsatellites?

Back 37

Replication slippage, caused by mismatches between DNA strands while being replicated during meiosis.·

Point mutations.

Front 38

What is one possible model of trint repeat expansions?

Back 38

Replication slippage model-

· Mismatches between DNA strands while being replicated during meiosis

· DNAP slips while moving along template strand and continue at wrong nt. More common when repetitive sequence is replicated.

Front 39

Explain Replication slippage model-

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· Tandem repeats and instability of sequence –loop out structures may form during replication while maintaining complementary base pairing.

· A nick one side of DNA strand is caused by cleavage by endonuclease – repetitive triplet is extending (DNAP) and sealed(ligase).

· Loop structure on parent strand – decrease no.of repeats; daughter – increase

Front 40

In Replication slippage model, what do most slippages result in ?

Back 40

Changes of one repeat unit, rates vary for different allele lengths and repeat unit sizes.

Front 41

When may there be increased instability during recombination?

Back 41

a large size difference between individual alleles,

Front 42

What is the FEN1 Okazaki fragment model?

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· Lagging strand replication - repeats form hairpin which are resistance to FEN1 endonuclease cleavage so DNAP re-associated incorrectly.

(Displacement of downstream Okazaki fragment to allow removal of 5’ overhangs by FEN1 endonuclease )

Front 43

What is the effect of expansion on mRNA

Back 43

Fragile X – autism.

Huntington’s

Front 44

Explain fragile X

Back 44

Normally, FMR1 has 5-60 CGG. If over 200 - fragile X.

In 5’ promoter sequence - methylation of CpG ina gene results in silencing of transcription. Expansion of the repeat - results in loss of function – no protein produced.

Variable expressivity

But anticipation and X-inactivation

Front 45

Explain Huntington's

Back 45

· Normal transcription and translation but leads to abnormally long version of protein. The elongated protein is cut into smaller, toxic fragments that bind together and accumulate in neurons,disrupting the normal functions of these cells.·

Front 46

Molecular analysis of triplet repeat expansion

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· Southern blot hybridisation – good at picking up large DNA fragments. Con: requires probe to gene where expansion is.

Front 47

Another method for detecting triplet repeat expansions?

Back 47

Triplet Repeat- Primed PCR (TP-PCR)

Front 48

Explain Triplet Repeat- Primed PCR (TP-PCR)

Back 48

· Design PCR primers. Reverse primer designed to3’ end of CGG repeats.

· R primer anneals randomly across CGG region during the PCR reaction

· Different sized PCR products produced

· Capillary electrophoresis produces a stutter pattern – separates fragments by size.

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Applications

Back 49

· Used in DNA profiling in kinship analysis and in forensic identification

· Amplified by PCR, using the unique sequences of flanking regions as primers

· Repetitive DNA not easily analysed by next genDNA sequencing, which struggle with homo polymeric tracts. So, analysed by conventional PCR amplification followed by Sanger sequencing.

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Use in forensic analysis

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· Tetra- or penta nt repeats are used – give a high degree of error-free data while being robust enough to survive degradation innon-ideal conditions.

· Longer repeats - suffer more highly from environmental degradation and do not amplify by PCR as well as shorter sequences

· Amplify by PCR, electrophoresis – determines howmany repeats they are. Visualised with fluorescent dyes – highly sensitive,safe, and expensive.

Front 51

What is the main effect of expanded repeats?

Back 51

Gain of function

Front 52

Microarrays - priniciple

Back 52

hydrogen bonds between complementary nucleotide base pairs

high number of complementary base pairs in a nucleotide sequence means tighter non-covalent bonding between the two strands.

Wash

cDNA labellign- Cy3 and Cy5