DNA2 Flashcards
what molecular causes are there of aneuploidy (missegregation)?
- centrosome number (dependent on growth signalling pathway) - cells with multiple centrosomes increases spindle attachments and missegregations rate2. chromosome cohesion - reduced cohesion (maintained by cohesin protein complex during G2 and M phases) increases missegregation3. organisation of spindle microtubules - incorrect kinetochore attachment4. 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 dup5. disruption of cell-cycle regulation could result in incorrect attachment of kinetochores
how can non-homologous repair mechanisms induce structural abnormalities?
NON-REPLICATIVE1. 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 translocations2. 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 translocations3. breakage fusion cycle - chromosome instability in cancerREPLICATIVE1. FoSTeS (fork stalling and template switching) 3’ lagging strand disenngages and anneals to ss DNA in nearby fork - causes dels, dups, inversions and translocations2. microhomology-mediated break-induced replication - restart of collapsed fork. leads to complex chromosome rearrangements3. 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
what is the biggest cause of recurrent chromosomal rearrangements?
NAHR between low copy repeats or SINEs, LINEs
what causes reciprocal translocations?
NAME?
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
what influences reciprocal translocation partner?
proximity in nucleus, frequency of DS breaks, fragile site
what are the Theoretical mechanisms of formation of robertsonian translocation?
involves only chromosomes 13, 14, 15, 21 and 221. centric fusion of acrocentrics2. break in one short arm and one long arm3. break in both short arms and formation of dicentric4. misdivision of centromere5. U type exchange (chromatids break and loop to join each other)6. isochromosomeoccur in 1% of recurrent pregnancy loss patients and 3% of infertile men. 75% of robertsonian translocations involve chr 13 and 14
what are terminal deletions and how are they stabilised?
- caused by DSBs and stabilised by1. synthesis of new telomere 2. obtaining telomere sequence from another chromosome3. chromosome circularization leading to ring chromosomerepetitive elements Alu, LINE, SINE and long terminal repeats LTRs play a role
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)
how are isochromosomes formed?
- misdivision of centromere2. U-type exchange
what is the most common structural abnormality and incidence?
balanced translocation 1:500
what are the different modes of segregation for balanced translocation?
2:2 (6 outcomes)alternate = normal or balancedadjascent 1 - non homologous centromeres travel togetheradjacent 2 - homologous centromeres travel together3:1 (8 outcomes)tertiary - 2 normal, 1 derivative AND 1 derivative monosomyinterchange - 2 derivatives, 1 normal AND 1 normal monosomy4:0 (unviable) (2 outcomes)
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
what are the different modes of segregation for Heterologous (different chromosomes) robertsonian translocations? which may be viable?
2:1 alternate = normal or balancedadjascent = disomic or nullosomic3:0 - 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)
what are outcomes for Homologous robertsonian segregation?
NAME?
how do inversions behave at meiosis?
- inversion loopPericentric = cross-over outside inversion gives normal or balanced gametes- crossover within inversion gives normal, balanced and unbalanced gametes2. paracentric = outside loop gives normal or balanced gameteswithin loop gives normal, balanced and unbalancedAll recombination products are dicentric or acentric and usually lost or not viable
how do insertions behave at meiosis?
INTERCHROMOSOMAL - up to 50% viability1. independent synapsing - insertional segment loops out on donor and recipient2. forms quadrivalent (if larger) and recombinant chromosomes with normal, balanced, del, dupINTRACHROMOSOMAL - risk higher for smaller segments- looping out most likely and odd number of crossovers results in recombinant chromosomes
what is a pericentric insertion?
between arms
what is a paracentric insertion?
within arm
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% sporadicexpect symmetric segregation but dynamic mosaicism may occur (daughters partially or totally aneuploid)
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 meiosissmall markers prone to loss at meiosis- may interfere with segregation causing infertility <1% recurrenceknown examples = isodicentric 15, inversion dup 15, i(12p) pallister killian syndrome
what is an isochromosome?
mirror image with identical arms- may be isodicentricmay present as supernumerary eg. pallister killian- usually de novo
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)
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
what abnormalities can NAHR cause?
NAME?
what two molecular mechanisms cause NAHR?
NAME?
what are Paralogous genomic segments?
Non-allelic genomic segments with identical DNA sequence, typically hundreds of kilobases in size and flanking breakpoint junctions.
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
Give Examples of known LCR/NAHR mediated deletions and duplication syndromes?
- PWAS - 4MB del 15q11-q13 with large cluster of complex repeats BP1-BP42. CMT1A/HNPP 17p12 encompassing PMP22 gene3. NF1 1.5MB del of NF1 gene on 17q11.2 mediated by 85kb LCR containing several genes and pseudogenes4. Di george 22q11.2 deletion
define mosaicism
as the presence of two or more genetically different cell lineages within one individual that have arisen in a single zygote
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
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)
why is identification of mosaicism in Turner syndrome important?
NAME?
what is the karyotype for Kleinfelter? when might they be fertile?
XXY mosaic 47,XXY/46,XY chromosome complement may be fertile
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
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 CNVcan be limited to somatic cells or include germline
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
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
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
what % of FRAX males ae mosaic?
20% for full mutation and premutation due to somatic instability
what is pseudomosaicism
mosaicism arising in culture
what is chimerism?
• Presence in an individual of two or more different cell lines derived from different zygotes
what level of mosaicism does karyotype and FISH detect?
8% at 0.9 confidence and 15% at 0.99 confidence
what level of mosaicism does microarrays detect?
• aCGH: detect > 10%.• SNP: detect <5%
what level of mosaicism does MLPA detect?
30-40 %. dups harder to detect than dels
what level of mosaicism does sanger detect?
10-20 % duplications harder to detect
what level of mosaicism does NGS detect?
1% - needs 5000x coverage
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.
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
name a MeCpG binding protein implicated in disease?
MECP2 rett syndrome (X linked)
what mechanisms are there for epigenetic gene regulation?
1) DNA methylation2) histone modification3) Non-coding RNA
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
what are the two main ways histones can be modified
- 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 = silencing2. methylation/demethylation - adds or removes methyl groups from free amino groups of lysines or arginines. catalysed by methyltransferases or demethylases. Effect is gene dependentalso phosphorylation and ubiquitination
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
when a gene is switched off, is chromatin open or closed, methylated or unmethylated cytosines and acetylated or deacetylated histones?
closed, methylated, deacetylayed histones
how do non coding microRNAs decrease transcription in epigenetics?
- 22nt long- bind to 3’ UTR of target mRNAs inducing enzymatic degredation and preventing translation
why are microRNAs in thereapeutic use difficult?
multiple genes targeted by single microRNAone gene can also be targeted by multiple microRNAs
how do Long noncoding RNAs (lncRNAs) affecttranscription in epigenetics?
- 200bp long and regulate histone modifications and structural transformations that differentiate heterochromatin from euchromatin
ADD TO CARDS describe diseases associated with epigenetic defects?
- Rett syndrome XLD - neurodevelopmental disorder of females with arrested development 6-18 months. caused by LOF MECP2 gene which encodes a 5MeC- binding protein2. FRAX - >200 CGG repeats which result in CpG islands at promoter of FMR1 to be methylated which turns genes off and prevents production of FMRP3. 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 gene4. 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 genes6. Transient Neonatal Diabetes Mellitus (6q24 related) - IUGR, hypergylcaemia in neonate, paternal UPD6, paternal duplication 6q24,
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
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)-
how might methylation testing be used in PND? how is it done?
NAME?
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
how does epigenetic therapy work?
NAME?
what is a risk with epigenetic therapy?
NAME?
what is the XIST gene?
lncRNA coats X to be inactivated
when does x inactivation occur?
blastocyst days 5-6In subsequent mitoses this pattern of X inactivation is inherited stably by each daughter cell
what is a barr body?
inactive x in highly condensed heterochromatic state
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
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
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.
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
describe x linked conditions
- Rett MECP2 XLD de novo - neurodevelopmental disorder of females with arrested development 6-18 months and seizures. male lethality2. Incontinentia Pigmenti - NEMO de novo XLD - alopecia, neurological defects, females affected
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
what two methods can be used to detect % x inactivation?
- 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 calculated2. 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
what is UPD?
Two copies of a chromosome, or part of a chromosome, from one parent and no copies from the other parent
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)
what are the two causes of UPD?
non-disjunction in gametes or trisomy followed by chromosome loss
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
how does UPD affect genomic imprinting?
a. imprinting = Differential expression dependent on parent of originb. 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 parentd. UPD results in abnormal dosage of the imprinted gene products
how is UPD implicated in cancer?
NAME?
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
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 commonbecause of recombination in meiosis, a chromosome may be both isodisomy and heterodisomy. This is different to segmental UPD (part of chromosome)
what are the 3 categories of UPD?
- UPD for complete chromosome complement2. UPD of complete chromosome3. segmental UPD (11% of cases)
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
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 placentab) 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
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
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
in what scenarios is UPD testing recommended?
NAME?
what techniques are available to detect UPD?
- methylation-specific PCR2. MS_MLPA3. Bisulphite restriction analysis and PCR4. Methylation Sensitive (MS) melting curve analysis5. Pyrosequencing6. SNP array7. Southern Blotting using Methylation Specific (MS) restriction enzymes8. Microsatellite Analysis9. Whole exome/genome sequencing10. Cytogenetic analyses
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 sensitiveDISADVANTAGES - 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
describe how MS-MLPA works for UPD detect and advantages and disadvantages?
NAME?
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 bloodsDISADVANTAGES: 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
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.
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
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 inheritedAdvantages: 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.
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 peakADVANTAGES: : 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
describe SNP array use for UPD? what are advantages and disadvantages?
- ‚ñ™ SNP arrays are capable of detecting methylation differences across several CpG sites- ‚ñ™ 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
describe use of Whole exome/genome sequencing for UPD detection
- ‚ñ™ Long regions of homozygosity (ROH) can be identified through WES/WGS and resolved to UPD events - detection of stretches of loss of heterozygosity - ‚ñ™ 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- ‚ñ™ WES/WGS is especially powerful in detecting UPD which results in homozygosity for AR disease- ‚ñ™ 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 resultsNGS of bisulphite converted DNA is possible for targeted regions or at the genome-wide scale, although this is still largely within the research setting
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
why might unsynchronised cultures be set up?
urgent referrals needed in 48 hours - examine number and gross structural abnormalities
when can CVS be taken? what is spontaneous abortion risk?
11-12 weeks - earliest invasive prenatal sample<1%
when can amniocentesis be taken? what is spontaneous abortion risk?
between weeks 16 and 20 of pregnancy when it contains fetal cells<1%
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 abnormality2% risk
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
what samples can be used for prenatal back-up cultures?
CVS, amnio, fetal fluids or tissue (POC)
how many prenatal cell cultures should be set up?
- 3 independent cultures
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 mosaicismDigested 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.
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 incubatorBest practice involves use of both direct (straight from CVS) and long term cultures
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
what reagent doesn’t need to be added for unsynchronized harvesting?
thymidine - blocks cells in S phase for synchromisation
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
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
Name the 5 different forms of DNA which occur, depending on the hydration of the environment.
B-DNA - most common A-DNA Z-DNA Cruciforms H-DNA G4-DNA
How is chromatin structure maintained?
Cohesins, CTCF, and histones
What histone modifications are possible?
AcetylationPhosphyorlationMethylationUbiquitinationSumoylation
Name some proteins that are key architectural modellers associated with disease
SATBP1 - breast cancer prognosisCTCF - Silver-Russell and Beckwith WeidemannCohesin - Cornelia de LangeMLL2 - Acute leukaemiasMECP2 - Rett syndromeCREBBP - Rubenstein Taybi
What is the function of the centromere?
Chromosome segregation during mitosis and meiosis IISite of Kinetochore assembly
Describe the structure of the centromere
Alpha-satellite DNA repeats, constitutive heterochromatin, made of multiple centromeric proteins (CENPs)
Name two problems caused by centromere malfunction
Premature centromere division - age-depedent processPremature chromatid separation - diagnosed by puffing of the centromeres on Karyotype, cause of Roberts syndrome.
Name some diseases caused by telomere malfunction
Dyskeratosis congenitaCri-du-Chat - loss of hTERT gene
List the different types of chromosome banding techniques
R-bandingG-bandingDAPI/DA stainingC-bandingQ-bandingCD-bandingT-bandingG11-bandingReplication-banding (for SCE and chromosome breakage syndromes)NOR staining
Name some disorders associated with DNA replication problems
Dyskeratosis congenitaMeier-Gorlin syndrome - mutation in one of the ORC componentsHutchinson-Gilford ProgeriaBloom syndrome - mutation in BLM, a topoisomerase
Whcih DNA polymerase is associated with synthesis of the leading strand and DNA repair?
DNA polymerase delta.
In which stage of the mieosis does Crossing over occur?
Pachytene of meiosis I
In which stage of the cell cycle so oocytes get halted during foetal development?
Diplotene in meiosis I
How is splicing regulated?
Regulated by cis-acting and trans-acting elements. Regulation is cell and tissue-specific.Requires the presence of Exonic Splice Enhancer and Intronic Splice Enhancers.SR proteins are required as they bind he ESE/ISE and components of the spliceosome.Exonic Splice Supressors and Intronic Splice Supressors also exist.
How can splicing aberrations cause disease?
Mutations disruption of a splicing element - CancerGeneration of toxic RNA - Myotonic DystrophyMutations affecting a splicing factor - SMN1Overexpression of splice factors - Cancer
Name some therapies that target splicing mutations/aberrations
Antisense oligonucleotides - bind specific splicing targets and enhancing or repressing. Antisense oligos can also be used to target mutant transcripts.Trans-splicing - introduces artificial wildtype splice site into mutant sequence.
Name the three RNA surveillance mechanisms, and say what they detect.
- Nonsense mediated decay - premature stop mutation2. Non-stop mediated decay - STOP lost. extended 3’ sequence and long Poly-A tail3. No-go mediated decay - not well understood
How does the NMD pathway work?
Exon Junction Complexes attach to the exon-exon boundaries of the mRNA. The ribosome displaces EJCs as it traverses the transcript - if it detaches before all EJCs are removed the transcript is flagged for NMD.
Give an example of a target escaping NMD.
T cell receptor - NMD can be activated even if a premature STOP occurs within the last 50bp of the exon.
Give examples of how NMD is related to disease
NMD is triggered by frameshift mutations in dystrophin resulting in premature STOP codons. This results in haploinsufficiency. If the mutation is in-frame a much milder phenotype (Becker) is observed.Marfan Syndrome - NMD is correlated with disease severity.
How can NMD potentially be treated?
- Correct the premature STOP mutation - promote read through -CF, DMD, SMA.2. Eliminate the portion of the gene with the STOP mutation in it3. Inhibit the NMD pathway, e.g. SMG1 kinase inhibitors.
Name the 4 functions non-coding RNAs
rRNA - present in the NORs, transcribed as a single 13kb transcript. (Treacher-Collins)tRNA - transport the amino acid to the ribosome during translation. (MELAS, MERFF)snRNA - spliceosomal RNAs - two subclasses: Sm and Lsm. (SMN2, cancer, RP)snoRNA - RNA processing and modification (Cancer, PWS)
Name the 4 types of regulatory RNAs
miRNA - acts to regulate expession via the mRNA transcript - post-transcriptional gene silencingsiRNA - acts to regulate transcription via the DNA. Dicer-depedent.piRNA - regulate transposon activity (Infertility)lncRNA - involved in X-inactivation and imprinting, regulate transcription factor activation and alternative splicing.
What roles do ncRNAs play in disease diagnosis and treatment?
Diagnostic biomarkers of diseaseTherapeutic agents to control gene expression: MRX34 drug acts to repress oncogenes in the TP53 pathway.Antisense oligos could be a potential treatment for Angelman syndrome (Anti-UBE3A).
List the types of normal genomic variation
Heterochromatic variation (1qh, 9qh, 16qh, Yqh)CNV - 8p, near GTA4 region.LCRs - repeat tractsSNPsInversions (chromosome 9 inv common). Chr 11 inv Satellite DNA in centromeres and stalksFragile sites/repeat regionc - may be BrdU inducible. FRA11B predisposes to del 11q (Jacobsen syndrome).Variable numbers of tandem repeats.Microsatellite repeatsEpigenetics
What can cause DNA damage?
UV, free-radicals, radiation, chemicals, mutations in proof-reading machinery
What repair mechanisms do humans have?
MMR - MutS-Alpha and MutS-beta recruit the MutL complex - identify, and excise incorrect base, DNA ligase fills the gapBase Excision Repair - removed oxidative damageNucleotide Excision repair - removes UV damageDSB repair - Homologous Recombination Repair - RAD51 required suring S-phase to repair from sister chromatid. BRCA1/2 part of this pathwayDSB repair - NHEJ - no homology required, results in ligation of two non-homologous molecules, bases may be missing and leave a scar.DNA polymerase proof-reading and repairing.Gene conversionFoSTeSMMBIRNAHR
What are the five types or pseudogene?
Non-processedProcessed (reintroduced into the genome by reverse transcription)Unitary - deactivation of gene without duplicationRNA pseudogenesMitochondrial pseudogenes
Why are pseudogenes clinically relevant?
Gene conversion can lead to inactivation of active gene (e.g. PMS2)Homologous recombination can lead to del/dupDifficult to accurately sequence
Name some clinically relevant pseudogenes
SMA is caused by mutations in SMN1. It has a pseudogene (SMN2 - common mutations in exon 7). Recombination and gene conversion can result in loss of SMN1. However, if SMN2 is duplicated, this can restore some function and result in a less severe phenotype.CYP21A2 - CAH. Has a pseudogene CYP21A1PPKD1PMS2BRCA1
How can we get around the pseudogene problem in the lab?
Design primers specific to parent geneLong range PCR for parent gene then nested primers to sequencecDNA methods
How can dicentric chromosomes be created?
As a result of telomere erosion and breakage-fusion-bridge repair. May be the cause of chromosome instability in cancers
What is chromothripsis?
The joining, likely by NHEJ, of multiple fragments of chromosomes. This may cause the highly complex rearrangements seen in cancer cells.
Name the mechanisms via which reciprocal translocations can be formed?
NHEJ, FoSTeS, MMBIR, MMEJ.
What often mediates recurrent translocation?
LCRs and NAHR
Name some recurrent rearrangements
Prader-Willi/Angelman common deletion (3mb)Isodicentric 22q11.2 - cat-eye syndrome.AZFa/b/c deletion in male infertilityRobertsonian translocations
List the things that can go wrong during meiosis 1 to cause aneuploidy.
Premature homologue separationAnaphase lagNon-disjunctionRecombination failure - the chromosomes remain attached.
List the things that can go wrong in Meiosis 2 to cause aneuploidy.
Premature chromatid separationNon-disjunctionAnaphase lag
Define tertiary trisomy
The two normal chromosomes segregate with one derivative chromosome
Define interchange trisomy
Two derivative chromosomes segregate with a normal chromosome
What factors should be considered when estimating risk of recurrence of chromosome aneuploidy?
Location of imbalance and chromosome involved.If either parent has a balanced translocationHaploid Autsomal Length (basically, smaller chromosomes are more likely to be tolerated in imbalance than larger chromosomes as they contain a smaller proportion of the total genetic material)
What should you be particularly aware of when analysing a case with a robertsonian translocation involving chromosomes 14 and 15?
UPD.
How are inversions formed?
Inversion loops. Can be paracentric or pericentric. can result in loss/gain through recombination during meiosis 1
What is the Minimal Processing Segment?
The shorted section of homology needed for LCR-mediated NAHR.
What are the three forms of NAHR rearrangements?
IntrachromatidIntrachromosomalInterchromasomal
Do reciprocal deletions and duplications appear at the same rate?
No - deletions are more commonly detected. Possibly because they have a more severe phenotype.
Give an example of a reciprocal deleltion/duplication.
Potoki-Lupski syndrome and Smith-Magenis syndrome (17p11.2) - RAI1 geneDiGeorge syndrome and velocardiofacial syndrome (22q11.2)
Give some examples of recurrent deletions/duplications mediated by LCR/NAHR.
PWS/AS deletion - common deletion is 4mb, between BP1 or BP2 and BP3. Larger deletion extends to BP4.NF1 - common 1.5mb deletionDiGeorge syndrome and velocardiofacial syndrome (22q11.2) - common deletion between LCR22B and LCR22D; smaller deletion between LCR22A and LCR22B
List the types of mosaicism
SomaticGonadalSex chromosome (review Hook’s tables)Confined placental mosacismPseudomosaicismChimaerismMethylation/X-inactivationVanishing twinUPD
How are genes methylated; what proteins are involved?
DNMT1 - maintainsDNMT3A/B - de novo methylation
Which nucleotide is methylated for epigenetic modidfication?
Cytosine. Forms 5MeC
Give a brief overview on how imprints/methylation patterns are set
Gametogenesis - de novo methylation of genes in gametesEarly embryogenesis - genome-wide demethylationPost implantation - Large-scale de novo methlation by DNMT3A/B
Give some examples of diseases caused by aberrant epigenetic modification or imprinting.
FRAX - expansion in 5’ region of FMR1 becomes methylated and prevents gene expressionRett syndrome - duplication of MECP2Beckwith Weidemann syndrome and Russel-Silver syndromePW/AS
How is epigenetic modification involved in cancer?
Hypermethylation of the MLH1 promoter can cause Lynch syndrome as the tumour suppressor gene function is supressed
Give examples of Epigenetic therapies.
5-azacytodine inhibits DNA methylation to retain activity in otherwise inactivated genes.Drugs can also promote methylation.Need to be careful as any alterations to modification must be targeted.
What are the two forms of UPD? Describe the differences between each.
Uniparental Isodisomy - 2 identical homologues from one parent.Uniparental Heterodisomy - 2 different homologues from one parent.
What are the three types of UPD?
- Complete chromosomal UPD: all maternal = benign ovarian teratoma, all paternal = hydatidiform mole.2. Whole chromosome UPD3. Segmental UPD
How can each type of UPD arise?
- Complete chromosomal UPD - a disomic gamete joins with a nullisomic gamete2. Whole chromosome UPD - trisomy rescue, monosomy rescue
Why don’t you see reciprocal segmental matUPD in Beckwith Weideman syndrome?
Considered to be lethal. See only patUPD.
List the methods used to detect UPD.
Bisulphite modification and:1. msPCR2. Pyrosequencing3. msPCR and restriction digest4. SNP array5. QF-PCRNon-modified DNA:1. southern blotting - digest the sample with methylation sensitive enzymes to create bands on a gel.2. msMLPA - two tube reaction, digest one tube with methylation sensitive enzymes3. Whole Exome/Genome analysis
Described 4 methods that ASOs can be used to interfere with gene translation
ASO/RNA can bind to mRNA and flag transcript for degradation by RNase H. ASO/RNA can bind to a specific target (mutation site) on the RNA and flag for degradation. ASO/RNA can bind to the mRNA and prevent binding of the ribosome to disrupt translation ASO/RNA can bind to the mRNA at S/R protein sites (ESE) to cause exon skipping.
Bases - Purines/ Pyrimidines
Purines have 2 interlocked rings A and GPyrmidines have a single ring C and T
Number of hydrogen bonds?A-TG-C
G-C 3 hydrogen bondsA-T 2 hydrogen bonds
Why is RNA more unstable?
Additional hydroxyl at 2’ position
Nucleosome components
2 each of H2A, H2B, H3 and H4
Linker DNA histone
H1
Euchromatin is
Extended conformationTranscriptionally activeWeak binding of H1 histonesAcetylation of nucleosome histones
Heterochromatin is
CondensedNot expressedTight H1 binding
cis-acting splicing mutations
Within consensus donor/acceptorBranch point mutationsDisruption of cis-elements e.g. ESEs, binding of SR’s/hnRNPs
Major spliceosome molecules
U1, U2, U4, U5 and U6
Minor spliceosome molecules
U11, U12, U4atac, U5 and U6atac
Chromosome regions commonly showing variation in constitutive heterochromatin
1qh9qh16qhYqh
9q heterochromatin Inversions
inv(9)(p11q12) third heterochromatin in p arm (10%)inv(9)(p11q13) all heterochromatin in p arm (0.6%)
Mutations - mechanisms
- DNA damage - endogenous - internal chem events ( depurination, deanimation oxaditive damage) - exogenous - enviromental agents ( mutagenic chemicals, UV, ionizing radiation)2. Deficiencies in DNA replication/ recombination - uncorrected errors 1√ó10-4 - 10-6 mts/ gamete for given gene3. Defects in DNA repair
DNA repair mechanisms
- BER base excision repair2. NER nt excision repair3. MMR mismatch repair4. HR holomologous recombination repair5. NHEJ non-homologous end joining
Doxorubicin - how does it work?
Chemotherapeutic agent used in treatm of various cancers inc breast, bladder, ALLInhibits Topoisomerase II -> stops DNA replication, prevent further cell division > cell apoptosis
End-replication problem
Telomerase - reversed transcriptase (RNA dependent DNA polymerase);2 subunits : -TERT - protein subunit and -TERC -RNA subunit with tandem reapeat seq complim to telemere repeats; this provides template to extend the telomere
