01 - basics & techniques Flashcards
Name 3 genes involved in epigenetic/chromatin & disorders associated with them
1) Cohesion - Cornelia de lange
2) MLL (KMT2A) - Acute Leukaemia
3) MECP2 - Rett Syndrome
Name 2 disorders associated with telomere malfunction
1) Dyskaratosis Congenita
2) Cri-du-Chat (5p del) - involves the hTERT gene
Describe Bloom Syndrome. How do we test for it?
> Rare AR disorder
Primordial dwarfism, sensitive skin, characteristic facies
BLM gene
Mutations in BLM result in hyper-recombination between sister chromatids = increased SCE
Test: replication staining. Treat cells initially with thymidine and later switch to Brdu (thymidine analogue). Process for chromosome staining. Chromosomes which replicate early in S phase incorporate thymidine stain dark, whereas those replicating late, incorporate Brdu and appear light.
SCE visible as harlequin chromosomes. Count number of SCE
) x10 MORE SCE in Bloom syndrome
What genes are involved in G1 checkpoint, what is their role?
> Cyclins, CDKs, RB1, E2F
Cell cycle growth enables CDK-Cyclin D formation
leads to hyperphosphorylation of Rb1
Rb1 normal inhibition of E2F is removed
E2F is a transcription factor which is now able to promote expression of Cyclin E
Cyclin E - CDK2 formation can occur
Transition into S phase
Name 3 disorders due to defects in non-coding RNA
1) Schwachmann Diamond Syndrome (SDS) - affects many parts of body, notably Bone Marrow = reduction in white blood cell production (neutropenia). At risk of developing MDS (myelodysplastic syndrome)
2) MELAS - mutations in the mt-tRNA Leu (MTTL1) gene. mostly m.3243A>G
3) MERRF - mutations in the mt-tRNA Lys gene. mostly m.8344A>G
name 3 mechanisms of mutation generation & provide an example of each
1) DNA damage:
UV radiation can cause linked pyrimidines (C/T) - thymidine dimers
2) Defects in DNA repair:
Mismatch repair pathway (MMR)
Complexes Mutsa (MSH2/MSH6 heterodimer) and Mutsb (MSH2/MSH3 heterodimer) recognise mismatched bases and recruit MutL (MLH1) to excise the mismatched bases. DNA polymerase fills the gaps
3) Recombination errors:
NAHR - misalignment of homologous mediated by LCRs can result in deletions/duplication
name 4 mechanisms of DNA repair & associated disorders
1) Mismatch Repair (MMR):
- MSH2/MSH3 heterodimer) recognise mismatched bases and recruit MutL (MLH1) to excise the mismatched bases. DNA polymerase fills the gaps
- MSH2/MSH6/MLH1 associated with Lynch syndrome (HNPCC)
2) BER - base excision repair:
- removal of oxidative damage / damaged bases
- MUTYH mutations ass. with colon cancer
3) NER - nucleotide excision repair:
- removal of pyrimidine dimers (UV radiation)
- Xeroderma Pigmentosa (XP) & Cockayne Syndrome
4) dsDNA breaks (DSB):
- via Homologous recombination repair, which uses WT stand from homologue as template for repair.
- requires RAD51 part of the BRCA1/BRCA2 pathway
Provide 2 examples of disorders associated with pseudogenes
1) SMA (spinal muscular atrophy)
- AR disorder
- due to deletions or gene conversions (in 98%) of SMN1 gene. (2% due to SNVs in SMN1)
- SMN2 pseudogene exists, shares 98% homology with SMN1
- key difference is an ex7 variant which disrupts a putative ESE (exonic splice enhancer) resulting in aberrant SMN2 splicing.
- SMN2 does have partial function and so increased copies of SMN2 can ameliorate the impact of SMN1 lose
2) CAH - congenital adrenal hypoplasia:
- AR
- due to deletions / gene conversions / mutations in geees in the cortisol from cholesterol pathway - notably CYP21a2
- pseudogene CYP21A1P - 98% homology, but harbours many deleterious inactivating variants
- region the gene & pseudogene reside prone to recombination
- results in deletions or gene conversions (which introduces those pathogenic variants into the coding gene)
- can result in virilasation in females and lethal salt wasting
What genes are tested as part of ‘floppy baby’ pathway
SMA, DM1, PWS
list 4 erros which may occur during meiosis I or II which result in aneuploidy
1) Recombination failure - achiasmatic non-disjunction
homologues separate to same pole
2) Premature homologue seperation - loss of cohesion be sister chromatids (MI)
3) Premature Sister chromatid separation - loss of cohesion between sister chromatids
4) Anaphase lag - failure of chromosome to attach to spindle apparatus and so fails to be pulled to poles (lags behind) and can be lost outside re-forming nucleus
What are the 4 possible outcomes of a pachytene cross:
1) Alternative:
Alternative (diagonal opposites) segregate - forms NORMAL & BALANCED gametes
2) Adjacent 1:
non-homologous centromeres travel together = UNBALANCED
3) Adjacent 2:
homologous centromeres travel together = UNBALANCED
4) 3:1
tertiary trisomy, 2 normal, 1 derivative
What 2 techniques can be used to investigate X-inactivation
1) Cytogenetically - Replication banding:
- treat cells initially with thymidine, following by Brdu, so that late replicating (inactive X) incorporates BrdU which stains late under Giesma
2) Molecular - HUMARA assay:
- based upon PCR amplification of CAG repeats in the AR gene
- genomic DNA is digested by methylation specific DNA (cuts unmethylated DNA - so only the active, unmethylated X is cut)
- PCR primers designed to flank RE digest sites - so if digested, no PCR product
- compared digested Vs undigested PCR products in normal Vs Skewed control.
Provide 2 different sizing strategies
1) Electrophoresis (gel, polyaccridimide, capillary)
2) PCR based amplification (LR-PCR; TP-PCR)
What are the types of enrichment available for NGS. Given examples
1) PCR based.
- can be in house LR-PCR based amplification
- or a commercial multiple kits - TruSeq Nextera
2) Enrichment based.
- use RNA / cDNA oligo probes as bait. Design baits to ‘tile’ over a region of interest
- genomic DNA will need fragmenting (RE digest or sonication)
- examples: SureSelect, Haloplex
Provide 2 examples of 3rd generation sequencing technologies
1) SMRT - single molecular real time sequencing (PacBio)
- real time sequencing
- long DNA strands run through a polymerase physically tethered to bottom of a specialism nanowell
- fluorescently labelled dNTPs added - and light emitted upon incorporation by DNA polymerase
- detection achieved due to miniscule size of well - so small that laser light cannot pass entirely through - but stops at point polymerase is tethered
- slides have a lawn of these wells - thousands of sequencings reactions occurring simultaneously
- PROs: fast; long reads (CNVs, indels, inversions etc)
2) Nanopore:
- DNA strand passes through nanopore
- unique electrical signature of each base is measured as passes through pore
- PROs: very quick; hand held device
- CONs: high error rate
WGS is not currently considered a viable option for detection of triplet repeats (such as CAG associated with HD). What algorithm seeks to address this
Expansion Hunter (Dolzhenko et al 2017) shown to accurately detect expansions in the C9orf72 gene in over 200 patients with amyotrophic lateral sclerosis (ALS) at detection rate of 100% Furthermore it accurately predicted 99% of wild type patients correctly
List some advantages WGS has over ES
> Unbiased sequencing (compared to ES)
New disease causing genes discovered frequently (avg 12 a month)
Exome sequencing only covers approx 98% of the exome - and so 2% is missing. Studies have shown many missing exons are exon 1
Intronic and other non coding regions are increasingly shown to contribute to human disease
