Session 1: DNA and chromosome structure, function and gene discovery Flashcards
1
Q
what is a DNA molecule?
A
Polymer consisting of 5 C sugar deoxyribose, phosphate group & nitrogenous base (heterocyclic ring of C & N)
• Purines=A and G (2 interlocked rings) and Pyrimidines=C and T; a single ring
2
Q
describe the structure of DNA
A
sugar-phosphate backbone linked by phosphodiester bonds (3’ P links to 5’P)
N base links to 1’ C. base + sugar = NucleoSide
NucleoSide + P = NucleoTide
Double helix bound by hydrogen bonds between complimentary bases A:T (2H bonds) & G:C (3H bonds)
2 anti-parallel strands curve around each other resulting in major & minor groves
1 turn = 3.6nm
B (right-handed) DNA most abundant
3
Q
describe the structure of RNA
A
single-stranded
A pairs with Uracil
more unstable due to Additional hydroxyl group at the 2’ position
A-form helix
4
Q
how is DNA packaged
A
Nucleosome = 2nm DNA coiled around 8 +charged histones 2 x (H2A + H2B +H3 + H4)
Chromatosome = nucleosome + H1 (binds to linker DNA)
Nucleosomes joined by linker DNA
allows transcriptional activity
chromatin (30nm fiber) = consists of nucleosomes packed into a spiral of 6-8 nucleosomes per turn
Metaphase = DNA condensed to 1/10,000 (by topoisomerase II and condensins)
Interphase chromatin is varied in compaction level
5
Q
what are the 2 classes of heterochromatin?
A
Constitutive: condensed and generally inactive. Consists largely of repetitive DNA
Facultative: sometimes inactive (condensed) and sometimes active (decondensed) e.g. X-inactivation
6
Q
what is the function on nc-RNA
A
help regulate expression of other genes
7
Q
what is the Open Reading Frame?
A
sequence of nt triplets read as codons > amino acids. begin with initiation codon AUG methionine and ends with stop codon.
8
Q
what are regulatory factors? give examples of diseases
A
required by RNA polymerase to initiate gene transcription. cis- acting (same DNA molecule as genes they regulate) and trans-acting (produced by remote genes and migrate to site of action). Some genes occur in clusters regulated by a locus control region.
examples of diseases:
LDLR promoter variant causes FH
FMR1 5’UTR expansion causes gene methylation and promoter silencing in FRAX
9
Q
Describe cis acting regulatory factors:
A
cis = same DNA molecule as genes they regulate.
examples:
Promoter: regulator region 5’ end of gene to which RNA polymerase binds to initiate transcription. consists of core promoter (most proximal) - contains RNA polymerase binding site, TATA box (30bp upstream of mRNA start site) where transcription factors and histones bind, and/or initiator element (specified transcription initiation to RNA polymerase) and transcription start site. defines transcription direction
Enhancer: regulatory sequence that modulates rate of transcription in response to binding of activators. binding of regulatory proteins causes DNA between promoter and enhancer to loop out allowing interation of regulatory proteins with promoter TFs or RNA polymerase
silencer: repressors bind (inhibit activators) reducing transcription. prevent gene expression through cell-cycle
insulator: protects genes from inappropriate signals by blocking action of enhancer on promoter
10
Q
Describe trans acting regulatory factors:
A
trans = produced by remote genes and migrate to site of action:
transcription factors - controls rate of transcription by binding to specific DNA sequences
11
Q
Describe the 5’ UTR
A
Regulates translation. spans transcription start site (TSS) to nt before mRNA start site & binds ribosome for polypeptide synthesis. 20% of genes express alternate 5’ UTRs by using multiple promoters to regulate gene expression.
BRCA1 has 2 different transcripts derived from 2 different promoters which differ in 5’ UTRs. longer transcript predominantly expressed in breast cancers.
12
Q
describe the 3’ UTR
A
Regulates translation. immediately follows translation stop codon and contains terminator sequence (endpoint for transcription and releases RNA polymerase) and regulatory regions (control polyadenylation, translation efficiency, stability and gene expression)
13
Q
what is the polyadenylation signal
A
directs addition and cleavage of poly(A) tail to end of mRNA trancript - important for nuclear export, translation and mRNA stability. cleavage occurs 15-30 nts downstream from signal.
14
Q
describe the mitochondrial genome?
A
transmitted exclusively through females. DS circular molecule containing 37 genes coding for 2 robosomal. 22 tRNA and 13 polypeptides (subunits of enzyme complexes of oxidative phosphorylation system). heavy and light chain transcribed from different promoter regions in opposite directions. genes closely clustered and contain no introns.
15
Q
describe transcription?
A
5’ to 3’ synthesis of ssRNA by RNA polymerase II, complimentary to antisense DNA strand and same base sequence as sense strand (except T>U).
Initiation: TFs (trans-acting) bind promoter (cis-acting)& position RNA polymerase II for RNA synthesis.
RNA transcript undergoes splicing, capping and polyadenylation.
16
Q
describe 5’ capping
A
occurs shortly after transcription initiation. a methylated nucleoside is added to 5’ end of RNA via phosphodiester bond. protects from exonuclease activity, facilitates transport to cytoplasm, facilitates RNA splicing and attaches to ribosome during translation
17
Q
what is a ribosome?
A
RNA-protein complex composed of 60S subunit and 40S subunit
18
Q
what is tRNA?
A
30 different types. up to 95nts, translates mRNA>protein. anticodon loop recognises complimentary mRNA codon. the amino acid is covalently linked to 3’ OH group by tRNA synthetase. only first two bases fit base-pairing rules, the last base is a “wobble” base as genetic code is degenerate.
19
Q
describe the process of translation?
A
- initiation = 5’ cap of mRNA binds ribosomal small 40S subunit & scanned until start codon identified. initiator tRNA(met) pairs with AUG start codon and binds to P site of ribosome. binding of tRNA induces conformational change and transfer of peptide chain to A site occurs
- elongation: tRNA at A site moves to P site and used tRNA at P site then moves to E site and is released upon binding of next tRNA to to A site.
- termination = elogation ends with stop codon. no complimentary tRNAs so hydrolysis of bond between tRNA and polypeptide at P side occurs and the polypeptide is released
20
Q
give examples of errors in translation causing human disease
A
- BRCA1 longest 5’ UTR transcript expressed in cancerous tissue which is translated less efficiently so BRCA1 protein expression is inhibited in breast cancer tissue as opposed to normal tissue which contains both
- DM1 caused by triple expansion in 3’ UTR results in toxic GOF effect with translation dysregulation
- m.3243A>G tRNALeu(UUR) causes MELAS
21
Q
what are ncRNAs?
A
- • constitute the majority of the human transcribed genome (60%)
- not translated into proteins
- • participate in complex networks of interactions with other nucleic acids and proteins
- regulators of gene expression
- • involved in many biologic processes: cancer, inflammation, and neurologic diseases
22
Q
what are long nc RNAs?
A
- > 200 nt long
- > 20 000 have been identified
- not evolutionarily conserved
- similar to mRNA (often transcribed by RNA pol II, may be polyadenylated, can show complex splice patterns)
- several types: antisense, intronic, exonic, promoter-associated
- Biological processes: regulate gene expression in cis and trans, epigenetic, transcription, XI (XIST expressed in cis to silence x chromosome), genomic imprinting, RNA splicing
23
Q
give examples of how long nc RNA cause human disease?
A
- H19 involved in imprinting at 11p15 Beckwith-Wiedermann syndrome
- UBE3A - ATS (antisense transcription) in angelmann
- SMA-AS1 recruits chromatin-modifying complexes in SMA
24
Q
what is micro RNA?
A
- small nc RNA 22nt long
- highly conserved
- regulate gene expression by post-transcriptional gene silencing
- binds to 3’UTR of mRNA and either prevents translational machinery binding or promotes mRNA degradation through deadenylation of polyA tail
- involved in proliferation, apoptosis, differentiation and development
- resistant to RNases
25
what is si RNA?
- small ds nc RNA ~20 nt long
- downregulates expression of target genes
- processed from long ds RNAs (through dicer)
- can mediate gene silencing by direct heterochromatin formation
26
what is piwi interacting RNA (piRNA)?
- small nc RNA ~30nt long
- >23 000
- controls gene expression
- dicer-independent , expressed only in germline cells
- 3 groups: transposon deived, mRNA derived and lnc-RNA derived
- associate with PIWI proteins to create a silencing complex that induces degredation and methylation
27
what are circular RNA's?
- circular ss RNA molecules generated during back-splicing of mRNA to coavalently link 3' end of an exon to 5' end of upstream exon
- resistant to RNase digestion so more stable than linear RNA molecules
- regulate linear RNA transcription and protein production
- dysregulation implicated in cancer occurence and progression
28
why might nc RNAs be useful as diagnostic tools?
- aberrantly expressed in different conditions so can be used as early diagnostic/prognostic maker eg. colon, lung and breast cancer, myocardial infarction, heart failure, drug induced liver injury
- mi RNAs resistant to RNases
29
how can nc RNAs be used as therapeutic agents?
- use of siRNA for silencing a defective allele
-mi RNA profiling to identify drug responders in cancers
- exogenous siRNAs to modulate RNA alternative splicing & correct defective gene expression
- ncRNAs to regulate promoter activity
- majority of clinicial trials are focussed on miRNA signatures as biomarkers for diagnosis, prognosis or therapy response however toxicity issues and target specificity issues
- mi RNA therapeutics either restore miRNA function or inhibit miRNA function
30
what are antisense oligos?
ss nucleic acid sequences that target specific regions of pre-mRNA and modulate gene expression
31
give examples of antisense oligos used in gene therapy?
- SMA: spinraza (Nusinersen) modifies SMN splicing by blocking intron 7 splice site to include exon 7 in SMN2 transcripts resulting in more full length SMN protein. injected into spinal cord as cannot cross blood-brain barrier
- DMD - Exondys 51 - hybridises to exon 51 of DMD pre-MRNA causing it to be skipped during splicing which corrects the translational reading frame in certain DMD gene deletions resulting in shorter but functional protein
- HD - HTTRx suppress translation of HTT mRNA containing CAG expansion - targets HTT snps so doesnt target expansions in other genes
Angelmann - ASO against lncRNA UBE3A antisense transcript
32
what is a chromosome made of?
chromatin (DNA + protein)
33
what are the 3 types of chromosome structures?
1. metacentric = q and p arms equal length
2. submetacentric = arms unequal
3. acrocentric = very short p arms
autosomes numbered largest to smallest except chr21 smaller than 22
34
what is a centromere?
highly specialized chromatin provides foundation for kinetochore assembly (disc shaped protein structure) and site for sister chromatid attachment. sister chromatids remain attached until checkpoint is reached. attachment mediated by cohesin complex of proteins. as cell progresses to anaphase the cohesin is degraded allowing sister chromatids to be separated to opposite poles. most constricted region of mitotic chromosome
35
when does chromatid separation occur?
mitosis and meiosis II
36
describe diseases associated with centromere dysfunction?
1) premature centromere division (PCD) age dependent process occuring in women, leading to increase in x chromosome aneuploidy
2) premature chromatid separation (PCS) - separate chromatids and discernible centromeres and involves most chromosomes in a metaphase. 40% of normal individuals. heterozygous PCS = >5% of cells and may cause decreased fertility and increase in aneuploidy in offspring. AD BUB1B mutation
3) Roberts syndrome - chromosome breakage. LOF in ESCO2 (8p21.1) results in delayed cell division and incerased cell death. growth retardation, limb malformation, craniofacial, ID and cardiac abnormalities
37
what is the Kinetochore?
large multiprotein complex (>80) assembles on the centromere and acts as point of attachment for spindle. essential for segregation.
38
what is a neocentromere?
a new centromere that forms at an abnormal location
may form on acentric preventing them being lost during cell division. formed via two processes
1) inverted duplication of distal part of chromosome results in acentric marker
2) interstitial deletion forms ring and linear marker chromosome
neocentromere then formed which lacks repetitive a satellite DNA and CENP-B. associated with cancer and MR
39
what is a telomere?
- highly conserved gene-poor DNA-protein complex that cap the end of eukaryotic chromosomes and maintain normal structure.
40
what is the function of the telomere?
- protection during cell division
- maintain structure - prevents fusing with broken chromosomes, recombination or degredation
- important for chromosome positioning in chromosome pairing
41
what is the structure of telomeres?
-TTAGGG repeats(highly conserved) associated with telomere-binding proteins
-adjacent to this are subtelomeric repeats (not conserved)
- proximal to subtelomere repeats is chromosome-specific DNA and subtelomere
- 3' single stranded overhang 150-200 nt long can form telomeric loops to protect ends when replicating lagging strand
42
what is telomerase?
what is its structure?
what is its function?
- RNA-protein enzyme which extends synthesis of leading strand using reverse transcriptase.
- composed of two subunits TERT (protein) and TERC (RNA) consisting of antisense hexanucleotide sequence to TTAGGG telomere repeat
-acts as template to prime extension of telomeric sequence of the leading strand, providing a template for DNA polymerase to complete synthesis of the lagging strand
- prevents telomere shortening. relates to cell senescence and aging
43
give an example of a disease associated with telomere malfunction?
cri du chat 5p deletopn = cat-like-cry, microcephaly, palmar creases. deleted region included hTERT genetelomerase reverse transcriptases which maintains telomere.
44
what is the Nucleolar organizing region (NOR)?
- organizes nucleolus structure and contains 200 rRNA genes for protein synthesis
- positioned on short arms of acrocentrics
contains rRNA genes 5.8S 18S and 28S
- if active it stains darkly with silver nitrate (Ag-NOR staining)
45
what are replication origins?
- cis acting DNA sequences which bind proteins for DNA replication
46
what is a G-band evaluation score?
The minimum standard acceptable G band resolution for a given referral reason
47
what causes the banding pattern in Giemsa staining? what are the differences between G-dark and G-light stains?
- digest with protease (trypsin) and staining with Giemsa results in dark (heterochromatin) and light (euchromatin) bands.
- G-dark is AT rich, gene-poor, low histone acetylation level (lower transcription) and LINEs
-G light is GC rich, gene-rich, high histone acetylation level and SINEs
48
what is R banding?
- Reverse banding - euchromatin stains dark and vice versa - better for seeing telomeres as stained darker
49
what is Q banding?
- original banding method - heteromorphisms show differential intensity between homologues and individuals allowing identification of additional chromosomes and paternity studies (Y is most intense)
50
what is C-Banding
(Constitutive heterochromatin banding).centromeric material comprised of repetitive DNA, satellite DNA (short tandemly repeated sequences: Alpha-satellite DNA, DNA satellite some non-repetitive DNA). It is differentiated from facultative heterochromatin in that facultative heterochromatin is condensed only semi-permanently via epigenetic changes which are reversible and can allow DNA transcription. Constitutive heterochromatin is permanently untranscribable.
Constitutive heterochromatin is highly polymorphic, likely due to the instability of the satellite DNA. affects size and localisation of heterochromatin with no phenotypic effect. used to identify polymorphic variants in heterochromatic regions, inversions and rearrangements. useful to identify dicentric and pseudodicentric chromosomes and markers
51
what is T banding?
telomeres
52
what is a counter-stain?
Counterstaining is a technique that is used to induce banding with fluorochromes that bind and fluoresce uniformly throughout the chromosome. It is also used to enhance banding patterns that do not have a very high resolution. can stain 15p quick but fades quickly eg. DAPI
53
what is replication banding? why is it useful for bloom syndrome? what are features of bloom syndrome?
BrdU is incorporated into chromosomes which is a thymidine analogue. Stained and then the BrdU DNA is stains different colour. used to detect early and late replications, detect different cycles of replication and count sister chromatid exchanges. used in Bloom syndrome where SCE no longer prevented after DNA damage due to BLM mutations leading to hyper-recombination and 10x more SCE. Bloom syndrome features = sunlight sensitivity, dwarfism, immunodeficiency, azoospermia and POF
54
what is NOR staining?
- stains p arms dark of acrocentrics with actively transcribed rRNA genes. stained with silver nitrate (ag-NOR). uses: see if marker has satellites, to check maternity/paternity as staining pattern is heritable). only technique for staining satellite stalks, cheaper than FISH. BUT siddifult to G band afterwards and very messy
55
what is DNA replication? what are the three stages?
semi-conservative process where parental strands act as template for synthesis of new complementary strand. 3 phases:
1. Initiation: begins at origins of replication, recognised by the origins recognition complex (ORC) in S phase. Topoisomerases nick DNA to be unwound by helicases. Allows RNA primer to bind followed by polymerase. Primers provide 3' hydroxyl group for DNA polymerase to start synthesis.
2. Elongation - primers removed and replaced with nuclleotides and backbone sealed by DNA ligase. Two replication forks allows polymerase to move in opposite directions 5' to 3' adding dNTPs. Lagging strand is synthesized discontinuously - polymerase elongates a short stretch (okazaki) then moves to new primer as the helicase moves along.
3. Termination - RNA primers removed and gaps in okazaki fragments filled by polymerase D. Nicks are joined by DNA ligase completing fully replicated chromosome
56
what Genetic Diseases are related to DNA replication?
Bloom syndrome - BLM gene. AR disorder caused by a helicase defect. symptoms = sensitivity to sunlight, growth deficiency, predisposition to malignancy & chromosomal instability
POLE in somatic cancer- produces polymerase without proofreading ability found in CRC and endometrial cancer
57
what 3 conditions are required for DNA polymerase?
1. 5' to 3' direction
2. ss DNA only
3. needs free 3' end (provided by primase)
58
what is an end replication problem?
no template at end of chromosome for primase to copy to make the RNA primer for the lagging strand.
59
how is the end replication problem solved?
telomerase - reverse transcriptase has TERC subunit which is complimentary to telomere TTAGGG repeats. telomerase binds to 3' lagging strand and acts as template to extend telomere. lagging strand now has room for primase and can be extended. lagging strand however cannot be extended to extreme 5' end leaving an overhang which can form telomeric loop which protects telomere DNA from cellular mechanisms that repair ds-DNA breaks. In adult somatic cells, telomeric DNA does not get replicated and the telomeres shorten - aging.
60
what are the phases of the cell cycle? G0, G1, S, G2 and M?
G0 = resting phase
G1, S, G2 = interphase:
G1 = Growth phase where proteins and RNA made only. chromosome is a single double helix. At G1 checkpoint (restriction point) the cell is committed to division and moves to S phase
S= DNA synthesis replicates genetic material. each chromosome has 2 sister chromatids now.
G2 = cell continues growing.G2 checkpoint - ensures enough cytoplasmic material for mitosis and cytokinesis (cell division)
M = mitosis - cell stops growing. nuclear division and cytokinesis. Metaphase checkpoint M ensures cell is ready to complete division.
61
what are the stages of mitosis?
prophase = nuclear membrane breakdown, chromosomes condense and spindle fibers appear
Metaphase = align at centre
anaphase - centromeres divide & sister chromatids move to opposite poles
telophase = nuclear membranes formand chromosomes decondense + spindle disappears
cytokinesis = cytoplasm divides and parent has become 2 daughter cells with identical genetic info
62
how is the cell cycle controlled?
- cell cycle checkpoints - regulatory pathways that control order and timing. Regulated by heterodimeric protein kinases.
- Checkpoints are essential to ensure that the cell cycle halts if chromosomal DNA is damaged or critical processes such as DNA replication or alignment have not been completed properly.
- checkpoint G1/s (restriction checkpoint)
- G2 /m checkpoint
- M checkpoint
- TP53 plays role in G1/S and G2/M checkpoints. it is a critical component for DNA damage check and inhibits cell cycle progression
63
what are the steps for producing stained chromosome metaphase preparations FOR 72 hour synchronised culture in routine constitutional work ?
1. mitogens induce cell division of resting cells eg. PHA
2. synchronisation - inhibitors block cell cycle in S phase eg. thymidine
3. block released (washed out) after 16-22 hours so cells continue through G2 together
4. mitotic arrestants (colcemid) after 4.5 hours - stop cell division at metaphase and prevent spindle fibres forming
5. ADD HYPOTONIC SOLUTION EG. KCl WHICH INCREASES VOLUME OF CELLS GIVING CHROMOSOMES MORE SPACE TO SPREAD.
6. Fix cells with fixative eg. Methanol:Acetic Acid (3:1) which kills cells and prepares them for banding
slide making - drop of cell suspension added to slide. As the fixative evaporates it enlarges cell and flattens out onto slide surface.
Banding - slides aged in hot oven.
Hanks solution - ageing
trypsin - enzyme digests chromosome and allows staining
Leishman’s stain - colours light and dark
64
how does meiosis differ from mitosis?
two rounds of cell division to produce 4 daughter cells with half the number of chromosomes as original parent cell. daughter cells are not identical to parent cells unlike mitosis. MI = random independent assortment of chromosome pairs and crossover enables recombination. MII = separation of chromatids (to haploid state)
65
what are the stages of meiosis?
1. Prophase 1 - chromatin consenses, nuclear envelope breaks down, homologoues pair to form bivalents, crossing over between non-sister chromatids within homologue, homologues held by chiasmata
2. metaphase 1 - bivalents align along metaphase plate and spindle forms
3. Anaphase 1 - Homologous chromosomes drawn apart. Chromatids remain together
4. Telophase 1 - haploid daughters (in females secondary oocyte has more cytoplasm than first polar body)
5. Prophase/Metaphase II - nuclear envelope breaks down, new spindle and chromosomes (consisting of 2 chromatids align)
6. Anaphase II - centromeres separate and sister chromatids migrate to opposite poles
7. Telophase II - further cell division forms two haploid cells. in females you get a viable ovum and second polar body (non-viable)
66
when does crossing over occur? what happens ?
meiosis prophase I
two homologous chromosomes pair and equal exchange between two strands. sealed by DNA ligase
67
what are chiasma?
connection where crossing over occurs
68
what are potential negative consequences of recombination?
non-homologous crossover (high homology but are not alleles) - leads to loss or gain of material
single gene disorders caused by deletion or duplication of a single gene eg. BRCA1
contiguous gene disorders - several genes deleted or duplicated alters dosage
segmental aneuploidy syndromes eg. Di George 22q11.2, PWS
69
where does cell splicing occur?
nucleus
transcription > premRNA> processed in nucleus by 5' capping, poly Adenylation and splicing. splicing removess introns by endonucleolytic cleavage and ligation
70
what is the spliceosome?
A 60S complex involving five snRNAs and their associated proteins
71
how does the snRNA complex interact with pre-mRNA?
1. U1 snRNA binds donor site
2. U2 binds branch site and U1 and U2 join together to form lariat loop (transesterification reaction)
3. U4, 5 and 6 associate with U1 and 2 to form spliceosome
4. U5 binds both donor and acceptor sites and cleavage of acceptor site by transesterification joins exons together
5. Lariat intron & spliceosome unbound
72
how is splicing regulated?
cis and trans acting elements
73
what are the four classes of splicing regulatory elements?
1. exonic splice enhancers - Generally hexamers and are evolutionarily conserved. Interact with SR (Ser-Arg) proteins
2. exonic splice silencers - Variable sequences that bind heterogeneous nuclear ribonuclear proteins (hnRNPs)
3. intronic splice enhancers - Generally hexamers and are evolutionarily conserved. Interact with SR (Ser-Arg) proteins
4. intronic splice silencers - Variable sequences that bind heterogeneous nuclear ribonuclear proteins (hnRNPs)
74
what is an An exonic splicing silencer
ESSs (cis-regulatory element) inhibit or silence splicing of the pre-mRNA and contribute to constitutive and alternate splicing (exon skipping). It does this by interfering with core splicing complex eg. U1 and U2
75
what is an An exonic splicing enhancer ?
6 base DNA motif that enhances splicing - thought that they interact with U2 snRNA to do this. mutations in this sequence cause genetic disorders and some cancers
76
what is alternative splicing?
mechanism to regulate gene expression in eukaryotes. contributes to proteomic diversity because it allows for the generation of multiple proteins from a single gene. Most common is exon skipping. also alternative 5' or 3' splice sites, intron retention and mutually exclusive exons (different combinations of exons), alternative promoters and alternative polyA leading to proteins with different functions or activities
77
how can splicing defects impact on disease?
up to 50% of human disease mutations alter splice elements with 10% due to consensus splice mutations.
1. disruption of splicing elements - non-coding SNVs in BRCA, Ataxia telangiectasia, Retinitis pigmentosa. consensus change may cause exon skipping or intron inclusion. Branch site mutation = Ehlers-Danlos type II. disruption of cis-elements ESS/ESE eg. 3bp in-frame deletion in exon 3 of MLH1 causes disruption of an ESE and causes HNPCC. 1/4 of SNVs in exons 9 & 12 of CFTR result in abnormal splicing
2. toxic RNA eg. unstable repeat expansions cause dysregulation of alternative splicing such as DM 3' UTR CTG GOF or In DM2, CCUG GOF expansion affects the non-coding regions of the ZNF9 gene.
3. Mutations (trans-acting) affecting splicing factors - eg. ALS, DCM - inclusion of differentially expressed exons. CFTR intron 8 poly T and TG tracts cause exon-skipping. SMA - exon 7 SNV promotes exon skipping and production of a truncated, inactive protein.
78
what therapies are available to target splicing defects?
1. antisense oligos can be used to enhance or repress eg. blocking splice sites for exon skipping in DMD or ptromote exon 7 inclusion in SMN2. can also be used to target mutant isoform trancripts for degradation eg. CTG GOF repeat in DM1
79
what is no-Go decay?
mRNA transcripts on which ribosomes have stalled (e.g. due to secondary structures)
80
where in the gene escapes NMD?
last and 3' 50bp of penultimate exon and within first 100nt (uses alternate start codon)
81
describe mechanism for NMD?
exon junction complex proteins are normally stripped off mRNAs by translating ribosome. If a PTC is present it remains bound. once ribosome reaches PTC, the SURF complex is formed which interacts with EJC and NMD regulators to form mRNA decay-inducing complex (DECID) leading to mRNA degradation. OR premature release of ribosomes tags downstream mRNA for destruction
82
give examples of diseases caused by NMD?
DMD - out of frame mutation leads to mRNA degradation.
BRCA1 X mutations inactivate tumour suppression
ALS caused by FUS mutations leads to neuron death
83
what treatment is available forPTC? what issues are there?
read-through eg. Translana for DMD caused by nonsense variants
splice-switching oligos cause exon skipping eg. restore correct reading frame for DMD
NMD inhibitors - eg. increased expression of TP53
o Issues include delivery, toxicity, variability in cells, tissues, individuals
84
what is non stop-mediated decay?
Detection and decay of mRNA transcripts which lack a stop codon. May be due to premature 3’ adenylation where ribosomes translate into 3' region and stall and cannot eject the mRNA. Nonstop mediated decay mediates this problem by both freeing the stalled ribosomes and marking the nonstop mRNA for degradation in the cell by nucleases
85
how are premature stop codons distinguished from natural ones?
by exon junction complexes - if there are EJCs present downstream of mutant PTC this will trigger NMD
86
how does read-through gene therapy work?
tRNA with 2/3 complementary bases anneals to incorrect stop codon and is incorporated generating the full length peptide
87
what is ribosomal RNA? what disease is rRNA involved in?
a type of non-coding RNA which is the primary component of ribosomes (cytoplasmic and mitochondrial). it is bound to ribosomal proteins to form small and large ribosome subunits and is involved in translating mRNA into protein (via tRNA). Involved in Treacher Collins syndrome - mutations involved in ribogenesis
88
what are Small nuclear RNA and what disease are they involved in?
part of spliceosome complex, specifies where splicing occurs and involved in SMA-
89
what are Small nucleolar RNAs (snoRNAs) and what diseases are they involved in?
guide chemical modifications and processing of other RNAs. involved in Prader-willi syndrome - paternal loss of snoRNAs in SNRPN locus responsible for phenotypic features of PWS
90
what are tRNA's and what disorders are they involved in?
transfer the correct amino acid to the ribosome during protein synthesis. cytoplasmic and mitochondrial. Cytoplasmic has cloverleaf structure with 3 hairpin loops including the anticodon loop. Involved in the diseases MELAS (Mitochondrial encephalopathy, lactic acidosis and stroke-like episodes) caused by MTTL1 SNVs and MERRF (myoclonic epilepsy with ragged red fibers) m.8344A>G in mt-tRNALys gene.
91
what is tRNA wobble base pairing?
3rd base in anticodon can form H bond with several bases at 3' position of the codon
92
what is a heteromorphism?
normal variation of visible variant differing in size, morphology or staining properties (5% of human genome is structurally variable)
93
what types of normal variation is there in the human genome?
1. heteromorphism
2. recurrent balanced translocations
3. inversions - several pericentric classed as variants
4. supernumerary marker chromosome - many show karyotypic variation without phenotypic effect
5. Constitutive heterochromatic variation - AT rich, varies in length, satellitte DNA
6. acrocentric p arm
7. CNVs (>1kb) - dependent on gene content, breakpoints and insertion sites and regulatory elements. If >1%, inherited from normal parent more likely benign BUT may have variable penetrance and expressivity.
8. euchromatic variants - resemble duplications, but polymorphic.
9. repeat sequence polys
10. transposable elements - units of DNA that move within genome eg. LINES and SINEs (such as Alu element). may be associated with disease if disrupt coding region
11. satellite DNA/Variable Number Tandem Repeats (VNTRs)
12. mini and microsatellites
13. low copy repeats/segmental duplications - not associated with disease but NAHR may be pathogenic
14. epigenetic variation - methylation and acetylation can alter DNA structure and expression
15. fragile sites - uncoiled chromatin, low in gene content. can be silenced by hypermethylation
16. polymorphism eg. SNV. not stable over time
17. RFLPs - cleavage of DNA with restriction enzymes
18. ExAC/GnoMad, dbSNP. DGV, Decipher
94
by what 3 methods do mutations arise?
1. DNA damage
2. Recombination/replication errors
3. failure to repair eg. checkpoint errors
95
what is DNA damage ?
- endogenous eg. oxidative damage or exogenous eg. tobacco or UV
- causes mutations
96
what is DNA replication error?
DNA polymerase adds incorrect NT during replication and some mutations evade the 3’→5’ exonuclease “proofreading” enzyme. uncorrected errors range from 1 x 10-4 to 1 x 10-6 mutations/gamete for a given gene.
97
what DNA repair mechanisms exist?
1. direct reversal - enzymes reverse the chemical reaction eg. removal of methyl group
2. Excision repair - non defective strand used as template and damaged DNA is removed and replaced. Base excision repair, nucleotide excision repair and MMR
3. ds-break repair - joining free DNA ends (however may place oncogene next to promoter). Homologous recombination repair (HRR) and non-homologous end joining (NHEJ)
98
what is base excision repair?
- removes bases with oxidative damage. DNA glycosylases remove damaged base by cleaving N-glycosylic bond between target base and deoxyribose. This is then filled by DNA polymerase and sealed by DNA ligase.
99
what is Nucleotide excision repair (NER)?
what syndromes are associated with this system?
major cellular defence system against the carcinogenic effects of UV exposure that Removes pyrimidine dimers caused by UV radiation.
1. detects damage
2. nuclease excision of erroneous DNA section
3. fills gap with DNA polymerase
3. seals nick
Xeroderma pigmentosum (XP), Cockayne syndrome (CS)
100
what is mismatch repair?
- recognises mismatched bases incorporated during DNA replication and excises them, DNA polymerase fills gap, DNA ligase seals DNA
- MutSa and MutSb recognise mismatch and recruit MutL to excise bases
- defective MMR leads to replication slippage observed in microsatellites (microsatellite instability)- alterations in length of tandem repeats resulting in insertion/deletion loops during replication - common in cancers
eg. Lynch syndrome and MLH1 sporadic cancers
101
what is Homologous Recombination Repair (HRR)? what diseases are associated?
- repairs ds-breaks using sister chromatids as homologous templates during G2
- facilitated by BRCA1, BRCA2, RAD51
- HBOC, Bloom syndrome, ataxia telangiectasia
- NAHR - mediated between low copy number repeats and can be intra/inter-chromatid or interchromosomal eg. LCRs flanking NF1 or F8 (Haemophilia) and unequal crossover between non-allelic homologous regions
- gene conversion - non-reciprocal transfer between sequences eg. SMN
- also repairs collapsed or broken replication forks
102
what is Non-homologous Repair (NHR)?
Repairs DNA breaks without homologous template
Non-replicative mechanisms:
1) non-homologous end joining - ligation of two DSBs
2) brakage-fusion bridge cycle - sister chromatids that lack telomeres fuse to create dicentric chromosome. During anaphase they are pulled apart and cycle repeats
Replicative mechanisms:
1) replication slippage - trinucleotide or dinucleotide expansion during replication
2)fork stalling and template switching
3) c) Microhomology-mediated break induced replication (MMBIR)
103
what is a pseudogene?
A DNA sequence that resembles a gene but has been mutated into an inactive form over the course of evolution. It often lacks introns and other essential DNA sequences necessary for function.Group of long non-coding RNAs (lncRNA). They can positively or negatively regulate gene expression and are often aberrantly expressed in cancer
104
what are the 3 types of pseudogene?
1. processed (72% of pseudogenes) = derived from reverse transcription of mRNA, inactivate coding ability
2. unprocessed - segmental dups and defective
3. accumulation of mutations so loses coding capacity
105
Example of pseudogene as positive gene regulator on cancer?
PTENP1 - functions as tumour suppressor and upregulation causes growth inhibition of tumour cells. regulates parent gene PTEN. Implicated in breast cancer, melanoma
106
describe the SMN2 pseudogene
- non-processed centromeric pseudogene. high sequence homology to SMN1 - distinguished by single SNVs in exons 7 and 8 and 3 intronic. all are synonymous but the exon 7 SNV disrupts ESE leading to aberrant splicing of SMN2 exon7. SMN2 retains partial functionality. number of copies acts as disease modifier. increased SMN2 = milder and increased life expectancy. SMN1 can convert to SMN2.
107
what is the Chiasmata
Join bivalents together at locations along the length of the chromatids.
108
what is Cohesin
Joins sister chromatids together, and also helps to maintain chiasmata.
109
how can aneuploidy arise?
1. meiosis i) 1:1:1 or meiosis ii) 2:1
2. mitosis - somatic or acquired results in mosaicism. less frequent than meiotic and mostly due to anaphase lagging.
110
what molecular causes are there of aneuploidy (missegregation)?
1. centrosome number (dependent on growth signalling pathway) - cells with multiple centrosomes increases spindle attachments and missegregations rate
2. chromosome cohesion - reduced cohesion (maintained by cohesin protein complex during G2 and M phases) increases missegregation
3. organisation of spindle microtubules - incorrect kinetochore attachment
4. recombination problems at M1 eg. failure to establish chiasmata can lead to homologs segregating to same pole. paracentric inversion crossing over can lead to acentric fragment which is lost or pericentric inversion can lead to genetic imbalance with del or dup
5. disruption of cell-cycle regulation could result in incorrect attachment of kinetochores
111
how can non-homologous repair mechanisms induce structural abnormalities?
NON-REPLICATIVE
1. non-homologous end joining used to repair ds breaks can lead to dels or insertions at the breakpoints to make them compatible for joining - major mechanism for cancer translocations
2. microhomology-mediated end joining - ds DNA repair leading to dels and insertions at break sites that require short regions of homology - major mechanism for cancer translocations
3. breakage fusion cycle - chromosome instability in cancer
REPLICATIVE
1. FoSTeS (fork stalling and template switching) 3' lagging strand disenngages and anneals to ss DNA in nearby fork - causes dels, dups, inversions and translocations
2. microhomology-mediated break-induced replication - restart of collapsed fork. leads to complex chromosome rearrangements
3. Chromothripsis - genomic rearrangements that occur in a short time interval as a one-off event and the joining, possibly via NHEJ, of these remaining chromosome portions that have been shattered into hundreds of pieces
112
what is the biggest cause of recurrent chromosomal rearrangements?
NAHR between low copy repeats or SINEs, LINEs
113
what causes reciprocal translocations?
- formed via NHEJ, microhomology-mediated end joining, fork stalling template switching or microhomology-mediated break-induced replication
114
what is t(11;22)(q23.3;q11.2)? what syndrome can it give rise to in offspring?
most common recurrent translocation caused by similar palindromic repeats leading to intra-strand pairing. susceptible to DNA breakage and NHEJ. balanced carriers at risk of der(22)t(11;22) Emanuel syndrome as a result of 3:1 meiotic malsegregation - viable as derivative chromosome is small
115
what influences reciprocal translocation partner?
proximity in nucleus, frequency of DS breaks, fragile site
116
what are the Theoretical mechanisms of formation of robertsonian translocation?
involves only chromosomes 13, 14, 15, 21 and 22
1. centric fusion of acrocentrics
2. break in one short arm and one long arm
3. break in both short arms and formation of dicentric
4. misdivision of centromere
5. U type exchange (chromatids break and loop to join each other)
6. isochromosome
occur in 1% of recurrent pregnancy loss patients and 3% of infertile men. 75% of robertsonian translocations involve chr 13 and 14
117
what are terminal deletions and how are they stabilised?
- caused by DSBs and stabilised by
1. synthesis of new telomere
2. obtaining telomere sequence from another chromosome
3. chromosome circularization leading to ring chromosome
repetitive elements Alu, LINE, SINE and long terminal repeats LTRs play a role
118
how are ring chromosomes formed?
result from two terminal breaks in both chromosome arms followed by fusion of the broken ends or one end breaks and joins with opposite telomere. also formed by subtelmoeric fusion or telomere-telomere fusion with no deletion. Ring 22 repaired by NHEJ or Microhomology-mediated break induced replication (MMBIR)
119
how are isochromosomes formed?
1. misdivision of centromere
2. U-type exchange
120
what is the most common structural abnormality and incidence?
balanced translocation 1:500
121
what are the different modes of segregation for balanced translocation?
2:2 (6 outcomes)
alternate = normal or balanced
adjascent 1 - non homologous centromeres travel together
adjacent 2 - homologous centromeres travel together
3:1 (8 outcomes)
tertiary - 2 normal, 1 derivative AND 1 derivative monosomy
interchange - 2 derivatives, 1 normal AND 1 normal monosomy
4:0 (unviable) (2 outcomes)
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how would you assess the viability of a chromosomal imbalance?
- size of translocated segment
- literature
- higher risk if known syndromes eg. 13, 18, 21 or microdeletions eg. 1p36, wold hirschhorn or cri du chat
- consider UPD if known region eg. 7, 11, 14 or 15
- de novo apparently balanced translocations may have microdeletion at breakpoints, gene disruption, position effect
- haploid autosomal length - 2% monosomy and 4% trisomy may be viable
- In females X;autosome translocation more likely to be viable as can inactivate the X chr
- In males, X'autosome translocation causes spermatogenic arrest
123
what are the different modes of segregation for Heterologous (different chromosomes) robertsonian translocations? which may be viable?
2:1 alternate = normal or balanced
adjascent = disomic or nullosomic
3:0 - very rare
- chromosomes 13 or 21 may produce viable offspring with Patau or DOwn syndrome
- chromosomes 14 or 15 could lead to offspring with UPD syndrome (after post-zygotic correction)
124
what are outcomes for Homologous robertsonian segregation?
-only disomic gamete or nullisomic gamete
- may have normal child if trisomic correction and no UPD associated
- monosomic correction can also lead to UPD but rare
125
how do inversions behave at meiosis?
1. inversion loop
Pericentric = cross-over outside inversion gives normal or balanced gametes
- crossover within inversion gives normal, balanced and unbalanced gametes
2. paracentric = outside loop gives normal or balanced gametes
within loop gives normal, balanced and unbalanced
All recombination products are dicentric or acentric and usually lost or not viable
126
how do insertions behave at meiosis?
INTERCHROMOSOMAL - up to 50% viability
1. independent synapsing - insertional segment loops out on donor and recipient
2. forms quadrivalent (if larger) and recombinant chromosomes with normal, balanced, del, dup
INTRACHROMOSOMAL - risk higher for smaller segments
- looping out most likely and odd number of crossovers results in recombinant chromosomes
127
what is a pericentric insertion?
between arms
128
what is a paracentric insertion?
within arm
129
what is a ring chromosome? how might it behave at meiosis?
two breaks in one chromosome result in ends fusing to form a ring. 99% sporadic
expect symmetric segregation but dynamic mosaicism may occur (daughters partially or totally aneuploid)
130
what are Extra structurally abnormal chromosome (ESAC’s)? how can you assess pathogenicity?
• Supernumary chromosomes are structurally abnormal chromosome fragments that cannot be characterised fully by conventional techniques.
- pathogenicity depends on gene content
- if acrocentric short arms, typically harmless
- if larger with euchromatin, more likely pathogenic
- forms univalent at meiosis
small markers prone to loss at meiosis
- may interfere with segregation causing infertility
<1% recurrence
known examples = isodicentric 15, inversion dup 15, i(12p) pallister killian syndrome
131
what is an isochromosome?
mirror image with identical arms
- may be isodicentric
may present as supernumerary eg. pallister killian
- usually de novo
132
what are low copy repeats?
sequence elements with high homology that are common in the human genome. -
The locations of LCRs within the genome mean that some regions are by their nature more predisposed to being subject to aberrant recombination events than others.
- >1kb and have >90% sequence homology with reference genome, occuring in two or more genomic locations in tandem or interspersed.
- often located in pericentromeric and sub-telomeric regions .
- different from LINEs, SINEs and Alu repeats as not present in large numbers
- when two repeats are close, the region is a hotspot for crossovers as high sequence identity between repeats can lead to mispairing
- leads to recurrent genomic rearrangements (dels, dups, isodicentric, inversions)
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what factors influence the liklihood of NAHR?
- LCRs <10kb
- large repeat size
- high degree of homology
- distance between regions
- orientation with respect to each other
- Minimal Efficient Processing Segment - segments of minimal length sharing high similarity between low copy repeats
134
what abnormalities can NAHR cause?
- CNV in dosage sensitive gene
- gene disruption
- fusion genes
- position effects
- unmasking recessive traits
- communication interruption between alleles
135
what two molecular mechanisms cause NAHR?
- unequal crossing over (recombination)
- break-induced replication caused by fork stalling and template switching
136
what are Paralogous genomic segments?
Non-allelic genomic segments with identical DNA sequence, typically hundreds of kilobases in size and flanking breakpoint junctions.
137
what is FoSTeS (Fork stalling and template switching):?
- DNA replication fork stalls and lagging strand disengages from the original template and anneals to another replication fork nearby
- MECP2 duplication syndrome (Xq28), deletions and duplications of 17p13.3, deletions and duplications of 17p11.2p12, deletions and duplications in 9q34 and many others
138
Give Examples of known LCR/NAHR mediated deletions and duplication syndromes?
1. PWAS - 4MB del 15q11-q13 with large cluster of complex repeats BP1-BP4
2. CMT1A/HNPP 17p12 encompassing PMP22 gene
3. NF1 1.5MB del of NF1 gene on 17q11.2 mediated by 85kb LCR containing several genes and pseudogenes
4. Di george 22q11.2 deletion
139
define mosaicism
as the presence of two or more genetically different cell lineages within one individual that have arisen in a single zygote
140
for which autosomes are mosaic trisomies most common?
13 Patau syndrome - 5%
18 Edwards syndrome <5%
21 Down syndrome 2%
16 >1% of pregnancies and most commonly occurring trisomy which is always mosaic. mostly tissue-specific. IUGR and cardiac defects
141
how does mosaicism occur in Turner syndrome?
15% mosaicism, may be numberical eg. 45,X/46, XX or 45,x/46,xx/47,xxx or structural eg. 45,X/46,X,i(X)(q10) or 45,X/46,X,r(X)
142
why is identification of mosaicism in Turner syndrome important?
- implications for development
- numerical may have milder phenotype - taller, may enter puberty, may be fertile
- structural may have less of Turner stigmata
- individuals with markers must be investigated to determine if X or Y derived. If Y material present then risk of gonadoblastoma.
- If XIST is absent may have functional partial X disomy
143
what is the karyotype for Kleinfelter? when might they be fertile?
XXY
mosaic 47,XXY/46,XY chromosome complement may be fertile
144
give an example of a syndrome with tissue specific mosaicism?
• 12p Pallister Killian syndrome always present in a mosaic form
• Results in tetrasomy for the short arm of chromosome 12
abnormal cells significant levels in fibroblasts
145
give an example of a disorder caused by a mosaic point mutation?
NF1 - AD. some patients have segmental NF1 - localised to a single portion of body and results from pozy zygotic point mutation or CNV
can be limited to somatic cells or include germline
146
which mechanism causes the majority of mosaic aneuploidy?
post zygotic non-disjunction
- Non-disjunction can occur in an initially normal (46,N) zygote, with the generation of mosaicism for a trisomic and a concomitant monosomic cell line as well as a normal cell line
- Growth of the monosomic cell line is severely disadvantaged and may die out leaving normal and trisomic cell lines
147
what is trisomy rescue? What condition can this give rise to?
postzygotic correction of the aneuploidy , by anaphase lag (one of these homologues may be lost due to delayed movement of the chromosome during anaphase of mitosis. The chromosome fails to connect to the spindle or is drawn to the pole at too late a stage to be included in the reformation of the nuclear membrane. It then forms a micronucleus, which is lost0
- UPD
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what is Age Related Sex Chromosome Aneuploidy?
- loss of X is normal ageing process
>44 years, 14% of women have 1/15 cells with X chromosome gain and 21% have at least one cell with X loss
- Y loss is greater in older men
- Y loss is a common finding in bone marrow karyotypes
- loss of Y more likely in MDS, MPD or lymphoproliferative disease
149
what % of FRAX males ae mosaic?
20% for full mutation and premutation due to somatic instability
150
what is pseudomosaicism
mosaicism arising in culture
151
what is chimerism?
• Presence in an individual of two or more different cell lines derived from different zygotes
152
what level of mosaicism does karyotype and FISH detect?
8% at 0.9 confidence and 15% at 0.99 confidence
153
what level of mosaicism does microarrays detect?
• aCGH: detect > 10%.
• SNP: detect <5%
154
what level of mosaicism does MLPA detect?
30-40 %. dups harder to detect than dels
155
what level of mosaicism does sanger detect?
10-20 % duplications harder to detect
156
what level of mosaicism does NGS detect?
1% - needs 5000x coverage
157
what is epigenetics?
• Heritable and transient changes in gene expression that do not alter the primary DNA sequence
- contributes to variable expression in different cell types
- sustained by DNA methylation, histone modification and RNA-associated silencing
- epigenetic modifications responsible for X-inactivation and imprinting are heritable from cell to daughter cell, but not from parent to child.
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how are genes methylated?
addition of methyl CH3 to c5 of cytosine to form 5MeC
- almost entirely restricted to cytosines of CpG dinucleotides
- 70% of CpG dinucleotides are methylated
- carried out by DNMT DNA methyltransferase enzymes
- methyl group acts as a signal to MeCpG binding proteins (MBD1-4 and MECP2)
- concentrated on repetitive sequences eg. pericentric heterochromatin
- gene promoted CpG islands stay unmethylated
159
name a MeCpG binding protein implicated in disease?
MECP2 rett syndrome (X linked)
160
what mechanisms are there for epigenetic gene regulation?
1) DNA methylation
2) histone modification
3) Non-coding RNA
161
what is Histone modification?
- histones are the primary components of chromatin (DNA+protein complex) that make up chromosomes
- chemical modifications to histone tail amino acids determine chromatin conformation and DNA transcription
- in relaxed form it is active and can be transcribed
- in condensed form it is inactive and transcription doesn't occur
162
what are the two main ways histones can be modified
1. Acetylation/Deacetylation - adds acetyl group to free amino groups of lysines or arginines. catalysed by histone acetyltransferases and histone deacetylases. Lysine acetylation = transcription and deacetylation = silencing
2. methylation/demethylation - adds or removes methyl groups from free amino groups of lysines or arginines. catalysed by methyltransferases or demethylases. Effect is gene dependent
also phosphorylation and ubiquitination
163
when a gene is switched on, is chromatin open or closed, methylated or unmethylated cytosines and acetylated or deacetylated histones?
open, unmethylated and acetylated histones
164
when a gene is switched off, is chromatin open or closed, methylated or unmethylated cytosines and acetylated or deacetylated histones?
closed, methylated, deacetylayed histones
165
how do non coding microRNAs decrease transcription in epigenetics?
- 22nt long
- bind to 3' UTR of target mRNAs inducing enzymatic degredation and preventing translation
166
why are microRNAs in thereapeutic use difficult?
multiple genes targeted by single microRNA
one gene can also be targeted by multiple microRNAs
167
how do Long noncoding RNAs (lncRNAs) affecttranscription in epigenetics?
- 200bp long and regulate histone modifications and structural transformations that differentiate heterochromatin from euchromatin
168
ADD TO CARDS describe diseases associated with epigenetic defects?
- 1. Rett syndrome XLD - neurodevelopmental disorder of females with arrested development 6-18 months. caused by LOF MECP2 gene which encodes a 5MeC- binding protein
2. FRAX - >200 CGG repeats which result in CpG islands at promoter of FMR1 to be methylated which turns genes off and prevents production of FMRP
3. BWS - overgrowth, embryonal tumours, macroglossia. 11p15 abnormal methylation (loss if maternal methylation at IC2 or GOM at IC1), paternal UPD. LOF variants in maternal inherited CDKN1C gene
4. RS syndrome - small, macrocephaly - maternal UPD (loss of dad), paternal hypomethylation at IC1. Dels/dels/translocations at either 11p15 or 7q32 , maternal GOF variants in CDKN1C, paternal LOF variants in IGF2.
5. PW/AS hypotonia, LD, hyperphagia and sleep disturbance (PWS) or ataxia, epilepsy and hyperactive (AS) - 15q11-q13 mat UPD causes PWS and pat UPD causes AS. OCA2/UBE3A genes
6. Transient Neonatal Diabetes Mellitus (6q24 related) - IUGR, hypergylcaemia in neonate, paternal UPD6, paternal duplication 6q24,
169
what is the methylation status in cancer cells? what is it caused by and how does it affect gene expression?
- loss of DNA methylation in cancer cells,
- may be environmental or age related,
- causes high gene activation by alerting the arrangement of chromatin leading to genomic instability, reactivation of transposable elements and loss of imprinting
170
describe effects of loss of methylation in cancer cells?
- mitotic recombination leading to deletions, translocations
- loss of imprinting eg. IGF2 in BWS/RS
- disrupted imprinting associated with tumour formation
- CpG islands become exessively methylated switching genes off eg. TSGs such as MLH1, VHL
- hypermethylation of promoters can make a microsatellite unstable and lengthen or shorten it - MSI indicates a DNA repair gene defect eg. MLH1 (sporadic)
-
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how might methylation testing be used in PND? how is it done?
- imprinting disorder testing increasing for foetuses conceived by reproductive methods
- testing for FRAX and DM1
- MS-MLPA and methylation specific pyrosequencing with molecular karyotyping for UPD and CNVs
- in the future arrays and NGS will be implemented
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what are complications of methylation testing in CVS?
- mosaicism may result in false negative
- some CpGs of differentially methylated regions could be hypomethylated in CVS and amniocytes
- in frax, test after 12 weeks as not fully methylated.
- need to define sensitivity
173
how does epigenetic therapy work?
- ideal as it is reversible unlike DNA mutations
- most popular alters DNA methylation or histone acetylation
- methylation inhibitors reactivate silenced genes. act like cytosine and incorporates into DNA whilst replicating, drugs then block DNMT enzymes, approved for MDS
- Histone deacetylase (HDAC) - remove acetyl group to stop transcription
- HDAC inhibitors turns on gene expression > anti-tumour activity and neurodegenerative disease
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what is a risk with epigenetic therapy?
- epigenetic processes are widespread
- treatments must be selective to irregular cells, otherwise activating gene transcription in normal cells would make them cancerous
- however treatments look promising
175
what is the XIST gene?
lncRNA coats X to be inactivated
176
when does x inactivation occur?
blastocyst days 5-6
In subsequent mitoses this pattern of X inactivation is inherited stably by each daughter cell
177
what is a barr body?
inactive x in highly condensed heterochromatic state
178
how does x inactivation occur?
- (XIC) on Xq13acts in cis - long non coding RNA spreads along chromatin outwards resulting in deacetylation and methylation of histones and closed chromatin
- • The Tsix antisense transcript is transcribed from the antisense strand of XIST gene and represses
XIST
- • Once inactivation has occurred on one X, repression of the other XIST allele is maintained by methylation of its promoter by tsix
179
which regions on X and Y escape inactivation and why?
PAR1 and PAR2 on X and Y for gene dosage
- non pseudoautosomal XY homologous region where 15% of x linked genes are escape inactivation
- if additional X chromosomes are present, additional X's are inactivated but some genes are transcribed resulting in functional trisomy
180
what are the problems with an x;autosome translocatin?
-if the XIC is within the translocated segment, inactivation can spread into parts of the autosome.
-Ideally in a balanced t(X;A), the intact X is preferentially inactivated, and the two derivative chromosomes will together comprise the functional X. However, gene disruption at the translocation breakpoint may complicate a phenotype.
181
what is skewed x inactivation?
-ratio of >75%; extreme skewing at >90%
- increases with age
- • Random X inactivation means an X-linked dominant trait is usually less severe in a female carrier than in a male
- • A female heterozygote may show skewed X inactivation resulting in preferential inactivation of the X containing the pathogenic variant
• Heterozygous variants in Xist or Tsix can cause non-random X inactivation
- if there is no preferential x inactivation chance could lead to a female heterozygote for an X-linked trait showing a phenotype
- skewing may be different in different tissue types so blood may not be representative
182
describe x linked conditions
1. Rett MECP2 XLD de novo - neurodevelopmental disorder of females with arrested development 6-18 months and seizures. male lethality
2. Incontinentia Pigmenti - NEMO de novo XLD - alopecia, neurological defects, females affected
183
describe cytogenetic technique to identify which X is inactivated?
- BrdU incorporates uracil A-U bonds. UV treatment cleaves A-U bonds.
- inactive x is late replicating
-• Giemsa stain only binds to dsDNA, not ssDNA facilitating visual differentiation between early and late replicating chromatin
- in the active X, some parts replicate late and some early so some areas have T and some U resulting in a banding patterns whereas inactive X is all late replicating
- may be possible to detect if inactivation has spread to autosome
184
what two methods can be used to detect % x inactivation?
1. methylation-specific PCR - DNA modified with sodium bisulphite and unmethylated cytosine > uracil. PCR primers designed specific to methylated and unmethylated alleles allowing ratios to be calculated
2. HUMRA assay - polymorphic CAG repeat amplified in first exon of AR gene. DNA digested with methylation-specific enzyme that cuts unmethylated allele. PCR primers amplify the methylated allele. products separated by electrophoresis and patterns between digested and undigested alleles are compared
185
what is UPD?
Two copies of a chromosome, or part of a chromosome, from one parent and no copies from the other parent
186
what is the incidence of UPD?
1/3500 (Robinson et al., 2000, Liehr et al, 2010) or 1/2000 for all chromosomes -23andMe (more representative of healthy individuals in general population)
187
what are the two causes of UPD?
non-disjunction in gametes or trisomy followed by chromosome loss
188
when does UPD cause disease?
disrupts imprinting (chromosomes 6, 7, 11, 14, 15, 20), creates an autosomal recessive condition on a chromosome not subject to imprinting (also x-linked disorder in females if there is UPD of the X chromosome).
cause of miscarriage if affects imprinted genes that control embryogenesis or activates recessive mutation for embryogenesis
189
how does UPD affect genomic imprinting?
a. imprinting = Differential expression dependent on parent of origin
b. Leads to monoallelic expression of either the maternal and paternal allele of a diploid locus (‘parent of origin effect’)
c. UPD for an imprinted region results in two active, expressed parental alleles or two silent, repressed parental alleles, depending on the contributing parent
d. UPD results in abnormal dosage of the imprinted gene products
190
how is UPD implicated in cancer?
- UPD resulting from somatic recombination can cause LOH or loss of imprinting eg. retinoblastoma, Wilms tumour.
- driver mutations become homozygous giving a clonal advantage
- somatic recombination leading to mosaic segmental UPD is common and results in late onset conditions
191
describe syndromes associated with UPD?
- paternal UPD6 = transient neonatal diabetes mellitus
- maternal UPD of 7 or 11 = silver-russell
- paternal UPD 11 = BWS
- mat and pat UPD 14 = temple syndrome and Kagami–Ogata syndrome
- PWS and AS mat UPD and pat UPD 15
- mat and pat UPD20 = Mulchandani–Bhoj–Conlin syndrome and pseudo-hypo-parathyroidism
phenotypic abnormalities of growth and behavior are common for UPD syndromes
192
what are the two types of UPD?
isodisomy = two identical copies of parental homologue (meiosis II nondisjunction)
heterodisomy = both homologues from one parent (Meiosis I nondisjunction). most common
because of recombination in meiosis, a chromosome may be both isodisomy and heterodisomy. This is different to segmental UPD (part of chromosome)
193
what are the 3 categories of UPD?
1. UPD for complete chromosome complement
2. UPD of complete chromosome
3. segmental UPD (11% of cases)
194
describe UPD for complete chromosome complement?
- Complete hydatidiform mole: UPD of entire diploid paternal chromosome complement 46, XX due to duplication of single 23, X sperm
- benign cystic ovarian teratoma: mat UPD for entire diploid complement - arises in egg due to failure of meiotic division
- triploidy partial hydatiform mole - extra set of chromosomes either maternal (digynic triploidy) or paternal (diandric triploidy) ussually paternal 69, XXY
- can get mosaic UPD/triploidy but rare
195
describe UPD of complete chromosome?
a) trisomy rescue - meiotic nondisjunction in one parent results in disomic gamete. fertilisation results in trisomic conceptus. posyzygotic mitotic nondisjunction results in a rescue through loss of one homologue or anaphase lag. most likely maternal heterodisomy from meiosis 1. mosaicism often observed with trisomic cell line confined to placenta
b) gamete complementation (rare) - meiotic nondisjunction in both parents results in disomic gamete from one parent and nullisomic gamete from the other for same chromosome. results in diploid zygote with UPD.
c) monosomic rescue (rarer than trisomic rescue) - nullisomic gamete leads to monosomic conceptus and rescue of remaining homologue results in UPD (isodisomy). may occur by pitotic nondisjunction, duplication or mitotic misdivision leading to isochromosome formation. most are paternal isodisomy.
d) post-fertilisation error - trisomy or monosomy rescue post-zygotically leads to both UPID
196
describe segmental UPD?
- UPD for part of one chromosome pair with biparental inheritance for the rest of the pair and caused by recombination e.g. Mosaicism for partial paternal isodisomy of 11p15.5 seen in 10-20% of cases with Beckwith Wiedemann syndrome
- segmental isodisomy formed postzygotically by mitotic exchange between non sister chromatids
197
in which scenarios may there be predisposition to UPD?
- robertsonian translocation carrier = translocation between two different homologous chromosomes - especially chromosomes 14 and 15
- reciprocal translocation at risk of 3:1 nondisjunction
- correction of monosomy
- isochromosomes = derived from single chromosome
- ring chromosomes may be corrected by compensatory UPD
- UPD may be associated with supernumerary chromosome
198
in what scenarios is UPD testing recommended?
- features consisted with imprinting syndrome
- familial chromosomal rearrangement involving imprinted chromosomes
- rare recessive disorder or unexplained transmission eg. homozygous child and het parent
- METHYLATION not set on CVS and placental methylation may not reflect fetal methylation
199
what techniques are available to detect UPD?
1. methylation-specific PCR
2. MS_MLPA
3. Bisulphite restriction analysis and PCR
4. Methylation Sensitive (MS) melting curve analysis
5. Pyrosequencing
6. SNP array
7. Southern Blotting using Methylation Specific (MS) restriction enzymes
8. Microsatellite Analysis
9. Whole exome/genome sequencing
10. Cytogenetic analyses
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how does sodium bisulphite PCR work for detecting UPD? what are advantages and disadvantages?
- Treating DNA with sodium bisulphite converts unmethylated cytosine nucleotides to uracil. Methylated cytosines (e.g. those in CpG islands of the methylated parental chromosome) remain unchanged.
- These differences can be used to investigate the methylation status of differentially methylated regions associated with imprinting using one set of primers specific for the treated methylated DNA sequence (for C's instead of U's), and one set specific for the treated unmethylated DNA sequence (for U's).
- resulting PCR products are run on a gel. As the products from treated methylated and unmethylated are of different sizes they will produce bands at different positions on the gel
- normal people have two bands and UPD has 1 band (same PCR product amplified from both chromosomes as same methylation status)
ADVANTAGES - does not require parental bloods. highly sensitive
DISADVANTAGES - cannot distinguish UPD or IC defect or segmental UPD. additional testing required to rule out a deletion (as still get one product from the one chromosome). PCR bias to unmethylated allele. high high false-positive rates - false-priming where amplification happens despite mismatches of primer with template and incomplete bisulphite treatment
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describe how MS-MLPA works for UPD detect and advantages and disadvantages?
- DNA denatured, MS-MLPA probes hybridised
- DNA mix split in two - one tube for standard mlpa copy number and the other is incubated with methylation-sensitive enzyme. probes ligated to unmethylated DNA are digested and not amplified. methylated DNA is not digested and will be amplified.
- copy number determinedwith undigested DNA and methylation pattern determined by comparing undigested sample to digested sample giving semi-quantitative methylation status.
- ADVANTAGES quick and cheap, doesnt require parental bloods, distinguish UPD/IC defect from a deletion, small amount of DNA, can detect duplications
- DISADVANTAGES - cannot distinguish UPD from IC defect
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how is Bisulphite restriction analysis and PCR used for UPD detection? advantages and disadvantages?
- bisulfite treatment, unmethylated DNA C>U
- produces different restriction sites on methylated vs unmethylated alleles
- region is amplified (outside treated area) and treated with restriction enzyme that cuts unmodified methylated DNA
- pcr products run on gel and different sizes for unmethylated vs methylated
- normal individuals have two bands. those with UPD have one band.
- ADVANTAGE - doesnt require parental bloods
DISADVANTAGES: cannot distinguish UPD from IC defect, additional testing maybe required to rule out a deletion. Also cannot distinguish segmental UPD. not all sequence changes caused by bisulphite modification result in formation or abolition of restriction enzyme site. Prone to heteroduplex formation not cleaved by restriction enzyme
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describe Methylation Sensitive (MS) melting curve analysis for UPD and advantages and disadvantages?
- bisulphite modification, PCR set up for fluorescent primers specific to methylated and unmethylated DN
- products are colled and then heated during which fluorescence in monitored
- unmethylated DNA has lower CG content and so melting temperature is lower
- two peaks for methylated and unmethylated products. UPD patients have one peak
- Advantages: Does not require parental bloods.
Disadvantages: This method cannot distinguish UPD from a deletion or IC defect – additional testing maybe required to rule out a deletion.
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describe Pyrosequencing for UPD analysis? advantages and disadvantages?
- Following treatment with bisulphite methylation differences can be detected and quantified by analysing the bisulphite-induced C/T differences at CpG sites.
- Pyrogram reports the ratio of cytosine to thymine at each site
- can be used to look at several CpG sites within a specific imprinting-related region, so in this sense is more targeted than SNP-array or whole exome/genome sequencing and can detect as little as 10% methylation
- high cycle numbers mean it is prone to contamination
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describe southern blotting using methylation specific restriction enzymes for UPD detection? what are the advantages and disadvantages?
- uses methylation sensitive restriction enzymes , which will not digest DNA with methyl group attached to cytosine.
- ▪ Using two restriction enzymes will therefore result in two different size fragments, a larger fragment for methylated DNA (cut by the standard enzyme only) and a smaller fragment for unmethylated DNA (cut by the standard and MS enzyme).
- ▪ Normal individuals will show 2 bands. Those with UPD will show only 1 band – which band is present will depend whether the mat or pat chromosome has been inherited
Advantages: Does not require parental bloods.
Disadvantages: Low throughput, poor sensitivity, requires large quantities of DNA.
This method cannot distinguish UPD/deletion or imprinting centre (IC) defect – additional testing maybe required to rule out a deletion. mosaicism difficult to assess and may have incomplete digestion.
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describe Microsatellite Analysis for UPD detection? advantages and disadvantages?
- Uses PCR to amplify DNA repeat sequences
- short tandem repeats (STRs). STRs are stable, short repetitive DNA sequences that are comprised of repeated elements. They are highly polymorphic and vary in length between individuals depending on the number of repeats.
- - Fluorescently tagged primers for microsatellites within the chromosome of interest are amplified and quantified using QF-PCR
- - Parental bloods are also analysed to determine the inheritance of the microsatellites in the patient
- Normal individuals who are heterozygous for a polymorphic repeat will show two peaks of the same height, a normal homozygous result will show one peak
ADVANTAGES: : Can potentially detect deletions, UPD and segmental UPD. Can distinguish hetero and isodisomy (peak looks twice as high homozygous in parent and child)
DISADVANTAGE: Requires parental bloods, markers may not be informative
207
describe SNP array use for UPD? what are advantages and disadvantages?
- ▪ SNP arrays are capable of detecting methylation differences across several CpG sites
- ▪ UPD can be detected by using homozygosity profiling with a SNP array (although homozygosity will flag regions of isodisomy, but not heterodisomy, if parents are heterozygotes). If parents are homozygotes, SNP array testing cannot distinguish between isodisomy and heterodisomy. This method therefore relies on testing large numbers of SNPs to maximize how many are informative
- Advantages: Can detect deletions, UPD, segmental UPD, hetero/isodisomy (if the SNPs are informative). Genome-wide rather than targeted to a single imprinting-related region
- Disadvantages: Requires parental bloods to detect UPD, expensive compared to targeted methods. Complete heterodisomy would be detected on SNP array only if trio genotype analysis was performed for all chromosomes, something that is not part of routine chromosomal microarray (CMA) analysis. A study of UPD detection by SNP microarray reported 10 of 30 confirmed UPD samples had no long contiguous stretches of homozygosity detected on the chromosome of interest, suggesting that up to one-third of whole-chromosome UPDs would not be detected by this method
208
describe use of Whole exome/genome sequencing for UPD detection
- ▪ Long regions of homozygosity (ROH) can be identified through WES/WGS and resolved to UPD events - detection of stretches of loss of heterozygosity
- ▪ SNP data also facilitates detection of mosaic UPD by detecting minor allele fractions with systematic departures from diploid genotypes (that are not associated with copy number change.
- bioinformatics allows for differentiation of biparentally inherited homozygosity and isodisomy, and also detection of heterodisomy
- ▪ WES/WGS is especially powerful in detecting UPD which results in homozygosity for AR disease
- ▪ Unlike SNP array which requires prior knowledge of the SNP locations, WES/WGS is a true exome/genome-wide approach. Parental samples are essential for interpretation of results
NGS of bisulphite converted DNA is possible for targeted regions or at the genome-wide scale, although this is still largely within the research setting
209
how can rt quantitative PCR be used for methylation detection?
- uses methylation and unmethylated-specific primers labelled with fluorophores to quantify degree of methylation in a sample
- can use blockers to increase sensitivity (blockers bind to unmethylated DNA, blocking access of primers so no PCR product generated).
ADVANTAGE: high throughput. false-positive rates are extremely low due to the blockers
DISADVANTAGE: cannot provide highly accurate quantitative methylation information about single CpGs within a region of interest as with Pyrosequencing
210
why might unsynchronised cultures be set up?
urgent referrals needed in 48 hours - examine number and gross structural abnormalities
211
when can CVS be taken? what is spontaneous abortion risk?
11-12 weeks - earliest invasive prenatal sample
<1%
212
when can amniocentesis be taken? what is spontaneous abortion risk?
between weeks 16 and 20 of pregnancy when it contains fetal cells
<1%
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when can fetal blood sampling be taken? what is spontaneous abortion risk?
from 17 weeks gestation - obtained directly from umbillical cord or fetus.
usually when CVS and amnio indicates an abnormality
2% risk
214
when can cff-DNA sampling be taken? what is spontaneous abortion risk?
- 5-7 weeks from maternal blood (only 6% is fetus)
- originates from placental trophoblast
- no abortion risk
- it is cleared rapidly after birth
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what samples can be used for prenatal back-up cultures?
CVS, amnio, fetal fluids or tissue (POC)
216
how many prenatal cell cultures should be set up?
- 3 independent cultures
217
why is testing of the cytotrophoblast cells in a CVS less likely representative of the fetus?
how can this be overcome?
more distantly related to the fetus than the mesodermal cells. abnormalities are more likely to be the result of confined placental mosaicism
Digested CV samples will contain a mixture of material derived from both cytotrophoblast and mesodermal cells. Long term culturing removes cytotrophoblast cells. If an abnormality is detected after digestion, long term culturing allows us to define if it is present in fetus or confined placental mosaicism.
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ADD TO CARDS how are CVS samples processed?
- CVS cleaned and maternal decidua removed
- villi transferred to culture media
- dipase to digest
- collagenase to suspend
- culture media wash to remove dipase and collagenase
- some suspension transferred for DNA extraction for array and molecular tests
- supernatant (liquid) removed from digest suspension and split into 3 independent cultures
- chang's media added (fetal bovine serum, antibiotics, glutamine and bicarbonate buffer for PH)
- placed in 37 degree incubator
Best practice involves use of both direct (straight from CVS) and long term cultures
219
how are AF samples processsed?
- portioned into extraction and cell culture
- aliquots centrifuged
- supernatant (excess liquid) removed
- pellet resuspended and wither transferred for DNA extraction or resuspended in Amniomax media (contains fetal bovine serum, glutamine, antibiotics and PH buffer)
- suspension examined under microscope to check for amniocytes and placed in incubator 37 degrees
- assessed a week later and media changed to ensure optimal conditions or if 5 or more colonies are present it can be harvested
- cystic fluid or fetal urine can also be processed
220
what reagent doesn't need to be added for unsynchronized harvesting?
thymidine - blocks cells in S phase for synchromisation
221
why is cell counting needed for cancer culture?
This allows for appropriate seeding of the culture (spread cells to a culture vessel ) so that the media is not exhausted of nutrients by over-seeding and enough cells are cultured for analysis in cases where a cell count is low
222
why don't cancer cells require PHA stimulation?
Neoplastic cells are already dividing
colcemid is often used as a mitotic arrest agent and the process of harvesing, slide making and G banding is the same for constitutional cell culture
