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
