Session 1 - Background

Question 1

Name the 5 different forms of DNA which occur, depending on the hydration of the environment.

Answer 1

B-DNA - most common A-DNA Z-DNA Cruciforms H-DNA G4-DNA

Question 2

How is chromatin structure maintained?

Answer 2

Cohesins, CTCF, and histones

Question 3

What histone modifications are possible?

Answer 3

Acetylation Phosphyorlation Methylation Ubiquitination Sumoylation

Question 4

Name some proteins that are key architectural modellers associated with disease

Answer 4

SATBP1 - breast cancer prognosis CTCF - Silver-Russell and Beckwith Weidemann Cohesin - Cornelia de Lange MLL2 - Acute leukaemias MECP2 - Rett syndrome CREBBP - Rubenstein Taybi

Question 5

What is the function of the centromere?

Answer 5

Chromosome segregation during mitosis and meiosis II Site of Kinetochore assembly

Question 6

Describe the structure of the centromere

Answer 6

Alpha-satellite DNA repeats, constitutive heterochromatin, made of multiple centromeric proteins (CENPs)

Question 7

Name two problems caused by centromere malfunction

Answer 7

Premature centromere division - age-depedent process Premature chromatid separation - diagnosed by puffing of the centromeres on Karyotype, cause of Roberts syndrome.

Question 8

Name some diseases caused by telomere malfunction

Answer 8

Dyskeratosis congenita Cri-du-Chat - loss of hTERT gene

Question 9

List the different types of chromosome banding techniques

Answer 9

R-banding G-banding DAPI/DA staining C-banding Q-banding CD-banding T-banding G11-banding Replication-banding (for SCE and chromosome breakage syndromes) NOR staining

Question 10

Name some disorders associated with DNA replication problems

Answer 10

Dyskeratosis congenita Meier-Gorlin syndrome - mutation in one of the ORC components Hutchinson-Gilford Progeria Bloom syndrome - mutation in BLM, a topoisomerase

Question 11

Whcih DNA polymerase is associated with synthesis of the leading strand and DNA repair?

Answer 11

DNA polymerase delta.

Question 12

In which stage of the mieosis does Crossing over occur?

Answer 12

Pachytene of meiosis I

Question 13

In which stage of the cell cycle so oocytes get halted during foetal development?

Answer 13

Diplotene in meiosis I

Question 14

How is splicing regulated?

Answer 14

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.

Question 15

How can splicing aberrations cause disease?

Answer 15

Mutations disruption of a splicing element - Cancer Generation of toxic RNA - Myotonic Dystrophy Mutations affecting a splicing factor - SMN1 Overexpression of splice factors - Cancer

Question 16

Name some therapies that target splicing mutations/aberrations

Answer 16

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.

Question 17

Name the three RNA surveillance mechanisms, and say what they detect.

Answer 17

1. Nonsense mediated decay - premature stop mutation 2. Non-stop mediated decay - STOP lost. extended 3’ sequence and long Poly-A tail 3. No-go mediated decay - not well understood

Question 18

How does the NMD pathway work?

Answer 18

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.

Question 19

Give an example of a target escaping NMD.

Answer 19

T cell receptor - NMD can be activated even if a premature STOP occurs within the last 50bp of the exon.

Question 20

Give examples of how NMD is related to disease

Answer 20

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.

Question 21

How can NMD potentially be treated?

Answer 21

1. Correct the premature STOP mutation - promote read through -CF, DMD, SMA. 2. Eliminate the portion of the gene with the STOP mutation in it 3. Inhibit the NMD pathway, e.g. SMG1 kinase inhibitors.

Question 22

Name the 4 functions non-coding RNAs

Answer 22

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)

Question 23

Name the 4 types of regulatory RNAs

Answer 23

miRNA - acts to regulate expession via the mRNA transcript - post-transcriptional gene silencing siRNA - 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.

Question 24

What roles do ncRNAs play in disease diagnosis and treatment?

Answer 24

Diagnostic biomarkers of disease Therapeutic 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).

Question 25

List the types of normal genomic variation

Answer 25

Heterochromatic variation (1qh, 9qh, 16qh, Yqh) CNV - 8p, near GTA4 region. LCRs - repeat tracts SNPs Inversions (chromosome 9 inv common). Chr 11 inv Satellite DNA in centromeres and stalks Fragile sites/repeat regionc - may be BrdU inducible. FRA11B predisposes to del 11q (Jacobsen syndrome). Variable numbers of tandem repeats. Microsatellite repeats Epigenetics

Question 26

What can cause DNA damage?

Answer 26

UV, free-radicals, radiation, chemicals, mutations in proof-reading machinery

Question 27

What repair mechanisms do humans have?

Answer 27

MMR - MutS-Alpha and MutS-beta recruit the MutL complex - identify, and excise incorrect base, DNA ligase fills the gap Base Excision Repair - removed oxidative damage Nucleotide Excision repair - removes UV damage DSB repair - Homologous Recombination Repair - RAD51 required suring S-phase to repair from sister chromatid. BRCA1/2 part of this pathway DSB 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 conversion FoSTeS MMBIR NAHR

Question 28

What are the five types or pseudogene?

Answer 28

Non-processed Processed (reintroduced into the genome by reverse transcription) Unitary - deactivation of gene without duplication RNA pseudogenes Mitochondrial pseudogenes

Question 29

Why are pseudogenes clinically relevant?

Answer 29

Gene conversion can lead to inactivation of active gene (e.g. PMS2) Homologous recombination can lead to del/dup Difficult to accurately sequence

Question 30

Name some clinically relevant pseudogenes

Answer 30

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 CYP21A1P PKD1 PMS2 BRCA1

Question 31

How can we get around the pseudogene problem in the lab?

Answer 31

Design primers specific to parent gene Long range PCR for parent gene then nested primers to sequence cDNA methods

Question 32

How can dicentric chromosomes be created?

Answer 32

As a result of telomere erosion and breakage-fusion-bridge repair. May be the cause of chromosome instability in cancers

Question 33

What is chromothripsis?

Answer 33

The joining, likely by NHEJ, of multiple fragments of chromosomes. This may cause the highly complex rearrangements seen in cancer cells.

Question 34

Name the mechanisms via which reciprocal translocations can be formed?

Answer 34

NHEJ, FoSTeS, MMBIR, MMEJ.

Question 35

What often mediates recurrent translocation?

Answer 35

LCRs and NAHR

Question 36

Name some recurrent rearrangements

Answer 36

Prader-Willi/Angelman common deletion (3mb) Isodicentric 22q11.2 - cat-eye syndrome. AZFa/b/c deletion in male infertility Robertsonian translocations

Question 37

List the things that can go wrong during meiosis 1 to cause aneuploidy.

Answer 37

Premature homologue separation Anaphase lag Non-disjunction Recombination failure - the chromosomes remain attached.

Question 38

List the things that can go wrong in Meiosis 2 to cause aneuploidy.

Answer 38

Premature chromatid separation Non-disjunction Anaphase lag

Question 39

Define tertiary trisomy

Answer 39

The two normal chromosomes segregate with one derivative chromosome

Question 40

Define interchange trisomy

Answer 40

Two derivative chromosomes segregate with a normal chromosome

Question 41

What factors should be considered when estimating risk of recurrence of chromosome aneuploidy?

Answer 41

Location of imbalance and chromosome involved. If either parent has a balanced translocation Haploid 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)

Question 42

What should you be particularly aware of when analysing a case with a robertsonian translocation involving chromosomes 14 and 15?

Answer 42

UPD.

Question 43

How are inversions formed?

Answer 43

Inversion loops. Can be paracentric or pericentric. can result in loss/gain through recombination during meiosis 1

Question 44

What is the Minimal Processing Segment?

Answer 44

The shorted section of homology needed for LCR-mediated NAHR.

Question 45

What are the three forms of NAHR rearrangements?

Answer 45

Intrachromatid Intrachromosomal Interchromasomal

Question 46

Do reciprocal deletions and duplications appear at the same rate?

Answer 46

No - deletions are more commonly detected. Possibly because they have a more severe phenotype.

Question 47

Give an example of a reciprocal deleltion/duplication.

Answer 47

Potoki-Lupski syndrome and Smith-Magenis syndrome (17p11.2) - RAI1 gene DiGeorge syndrome and velocardiofacial syndrome (22q11.2)

Question 48

Give some examples of recurrent deletions/duplications mediated by LCR/NAHR.

Answer 48

PWS/AS deletion - common deletion is 4mb, between BP1 or BP2 and BP3. Larger deletion extends to BP4. NF1 - common 1.5mb deletion DiGeorge syndrome and velocardiofacial syndrome (22q11.2) - common deletion between LCR22B and LCR22D; smaller deletion between LCR22A and LCR22B

Question 49

List the types of mosaicism

Answer 49

Somatic Gonadal Sex chromosome (review Hook’s tables) Confined placental mosacism Pseudomosaicism Chimaerism Methylation/X-inactivation Vanishing twin UPD

Question 50

How are genes methylated; what proteins are involved?

Answer 50

DNMT1 - maintains DNMT3A/B - de novo methylation

Question 51

Which nucleotide is methylated for epigenetic modidfication?

Answer 51

Cytosine. Forms 5MeC

Question 52

Give a brief overview on how imprints/methylation patterns are set

Answer 52

Gametogenesis - de novo methylation of genes in gametes Early embryogenesis - genome-wide demethylation Post implantation - Large-scale de novo methlation by DNMT3A/B

Question 53

Give some examples of diseases caused by aberrant epigenetic modification or imprinting.

Answer 53

FRAX - expansion in 5’ region of FMR1 becomes methylated and prevents gene expression Rett syndrome - duplication of MECP2 Beckwith Weidemann syndrome and Russel-Silver syndrome PW/AS

Question 54

How is epigenetic modification involved in cancer?

Answer 54

Hypermethylation of the MLH1 promoter can cause Lynch syndrome as the tumour suppressor gene function is supressed

Question 55

Give examples of Epigenetic therapies.

Answer 55

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.

Question 56

What are the two forms of UPD? Describe the differences between each.

Answer 56

Uniparental Isodisomy - 2 identical homologues from one parent. Uniparental Heterodisomy - 2 different homologues from one parent.

Question 57

What are the three types of UPD?

Answer 57

1. Complete chromosomal UPD: all maternal = benign ovarian teratoma, all paternal = hydatidiform mole. 2. Whole chromosome UPD 3. Segmental UPD

Question 58

How can each type of UPD arise?

Answer 58

1. Complete chromosomal UPD - a disomic gamete joins with a nullisomic gamete 2. Whole chromosome UPD - trisomy rescue, monosomy rescue

Question 59

Why don’t you see reciprocal segmental matUPD in Beckwith Weideman syndrome?

Answer 59

Considered to be lethal. See only patUPD.

Question 60

List the methods used to detect UPD.

Answer 60

Bisulphite modification and: 1. msPCR 2. Pyrosequencing 3. msPCR and restriction digest 4. SNP array 5. QF-PCR Non-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 enzymes 3. Whole Exome/Genome analysis

Question 61

Described 4 methods that ASOs can be used to interfere with gene translation

Answer 61

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.