Session 1: Basic Genetics

Question 1

Bases - Purines/ Pyrimidines

Answer 1

Purines have 2 interlocked rings A and G
Pyrmidines have a single ring C and T

Question 2

Number of hydrogen bonds?
A-T
G-C

Answer 2

G-C 3 hydrogen bonds
A-T 2 hydrogen bonds

Question 3

Why is RNA more unstable?

Answer 3

Additional hydroxyl at 2’ position

Question 4

Nucleosome components

Answer 4

2 each of H2A, H2B, H3 and H4

Question 5

Linker DNA histone

Answer 5

H1

Question 6

Euchromatin is…

Answer 6

Extended conformation
Transcriptionally active
Weak binding of H1 histones
Acetylation of nucleosome histones

Question 7

Heterochromatin is…

Answer 7

Condensed
Not expressed
Tight H1 binding

Question 8

cis-acting splicing mutations

Answer 8

Within consensus donor/acceptor
Branch point mutations
Disruption of cis-elements e.g. ESEs, binding of SR’s/hnRNPs

Question 9

Major spliceosome molecules

Answer 9

U1, U2, U4, U5 and U6

Question 10

Minor spliceosome molecules

Answer 10

U11, U12, U4atac, U5 and U6atac

Question 11

Chromosome regions commonly showing variation in constitutive heterochromatin

Answer 11

1qh
9qh
16qh
Yqh

Question 12

9q heterochromatin Inversions

Answer 12

inv(9)(p11q12) third heterochromatin in p arm (10%)
inv(9)(p11q13) all heterochromatin in p arm (0.6%)

Question 13

Mutations - mechanisms

Answer 13

1. 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 1×10-4 - 10-6 mts/ gamete for given gene
3. Defects in DNA repair

Question 14

DNA repair mechanisms

Answer 14

1. BER base excision repair
2. NER nt excision repair
3. MMR mismatch repair
4. HR holomologous recombination repair
5. NHEJ non-homologous end joining

Question 15

Doxorubicin - how does it work?

Answer 15

Chemotherapeutic agent used in treatm of various cancers inc breast, bladder, ALL
Inhibits Topoisomerase II -> stops DNA replication, prevent further cell division > cell apoptosis

Question 16

End-replication problem

Answer 16

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

Question 17

Chromatine proteins in disease

Answer 17

1. CTCF - chromatin loop eg. Silver Russell, Beckwith-Wiedemann Syndrome
2. Cohesin - pairing of sister chromatids eg. Cornelia de Lange, Roberts Syndrome
3. C-MYC - maintain euchromatine open eg. various tumours
4. MLL - inv in early develop and hematopoiesis eg acute leukemias, poor prognosis
5. MECP2 Rett Syndrome
6. CREBBP Rubinstain -Taybi Syndrome

Question 18

Telomere function

Answer 18

1. Maintain structural integrity – if lost the chromosome end is unstable (becomes ‘sticky’);
2. Prevents shortening of the chromosomes at each round of cell division.
3. help to establish the 3-D architecture of the nucleus and aid chromosome pairing.

Question 19

Telomeropathies

Answer 19

· Cri du Chat syndrome (CdCS)
· Dyskeratosis congenital (DC)
· Anaplastic anaemia

Question 20

Splicing regulation

Answer 20

Regulation
in tissue (cell chemistry),
context (interaction with other trans-acting elements),
timing (cell cycle)

Splicing regulatory elements: ESS, ESE, ISS, ISE - can be cis-/trans- acting

Question 21

Alternative splicing - types

Answer 21

1. exon skipping
2. altern 5’ splice site
3. altern 3’ splice site
4. intron retention
5. mutually exclusive exons

Question 22

Altern splice proteins - examples

Answer 22

1. WT1 - 4 isoforms: +/- KTS isoforms (3AA in ex9), +/-17aa (in ex 5)
2. Calcitonin (CGRP) - tissue specific splicing
   ex 1-4 - thyroid C cells
   ex 1-3, 5-6 - neurons
3. Tau (MAPT) - 3R/4R tau isoforms (alter splicing of ex 10) > FTDP-17

Question 23

Mutations within consensus splice sites account for (%)

Answer 23

10% of all disease causing mutations

Question 24

Splicing in disease - mechanisms

Answer 24

1. Disruption of splicing element (within +/-2bp, branch point, cis- elements) eg WT1 -KTS isoform, 3bp del in ex4 MLH1 > HNPCC
2. Toxic RNA - ^the stability of mRNA-RBP complex> repeat expansion eg DM1 CTG, DM2 CCTG
3. Mt affecting splicing factors eg TDP-43 (ALS), RBM20 (DCM)

Question 25

Founder mutation in FamDys

Answer 25

IKBKAP (ELP1) c.2204+6T>C (skipping of ex 20)

Question 26

Therapies targeting aberant splicing

Answer 26

1. GENE THERAPIES: - ASO targeting splicing signals ( to enhance/repress splicing) eg DMD - ASO for skipping ex 51 (Enterlipsen) SMA- ASO to promote SMN2 ex 7 inclusion (Spinraza) - ASO/ A/b targeting mutant isoform transcripts for degradation DM1 ARSE for (CUG)n repeat in RNA DMPK - SMaRT - post transcriptional correction of mRNA seq 2. molecules that promote/inhibit the activity of trans-acting factors that regulate alter splicing

Question 27

mRNA surveillance mechanisms

Answer 27

1. Nonsense Mediated mRNA decay pathway (NMD) - targeting transcripts containing premature termination codons (PTCs) 2. Nonstop Mediated mRNA decay pathways (NSD) - mRNA transcripts which lack a stop codon 3. No-go Mediated mRNA decay pathway (NGD) - mRNA transcripts on which ribosomes have stalled (e.g. due to secondary structures)

Question 28

NMD

Answer 28

predicted to occur if the PTC is upstream of the 3’ most 50-55 nucleotides of the penultimate exon (last exon-exon junction).

Question 29

Exceptions to NMD rule

Answer 29

1. T cell receptor gene rearrangements. Transcripts degraded by NMD despite having a PTC located within the last 50 nucleotides of the penultimate exon. 2. a long 3’ UTR can also target a transcript for NMD eg COL10A1 (mutations cause metaphyseal chondroplasia, type Schmid-MCDS). Mutation hotspot in large final exon 3- trigger NMD but deletion of conserved regions of the 3’UTR protects transcripts (with PTCs) from NMD 3. β-thalassaemia- transcripts containing nonsense mutations in 5’ region of exon 1 β-globin escape NMD

Question 30

NMD and disease

Answer 30

1. β-thalassaemia - premature stop in 1st or 2nd exon of three-exon β-globin gene. 2. DMD with disrupted reading frame 3. Marfan syndrome (FBN1) - dominant neg effect (nonsense mutations> low levels of mutant mRNA = mild phenotype ) 4. Cancer eg BRCA1, TP53 5. ALS (FUS)

Question 31

MELAS - most freq variant

Answer 31

m.3234A>G MT- TL1 80% cases

Question 32

MERRF most freq variant

Answer 32

m.8344A>G MT-TK 80 % cases

Question 33

At which stage of cell cycle recombination occurs

Answer 33

Pachytene - 3rd stage of prophase of meiosis I

Question 34

Decribe paracentric and pericentric inversion

Answer 34

PARACENTRIC - chr break and inv doesn't include the cenromere PERICENTRIC - includes centromere

Question 35

Homotrisomy

Answer 35

Occurence of more than one child with trisomy in the same family