Genetics

Question 1

CADD score

Answer 1

Combined annotation-dependent depletion score
- predicts pathogenicity (disease-causing potential) of variants/indels

Question 1

2 scores for predicting LoF constraint and their cut-offs

Answer 1

• pLI (probability of LoF Intolerant): =/> 0.9
• LOEUF (LoF observed/expected upper bound fraction): <0.35

Question 2

How to judge missense constraint on gnomAD

Answer 2

Using missense constraint Z-score >3.1

Question 3

Concept of “constraint” in genetics

Answer 3

Constraint describes how tolerant a gene is to genetic variation (different variants), ie a gene with high constraint is intolerant to variation.
E.g. LoF constraints (measured by pLI and LOEUF) and missense constraint (z-score)

Question 4

Concept of “depletion” and “enrichment” in genetics

Answer 4

Depletion/depleted: genetic variant observed as less common or less frequent than the expected value
Enriched: variant more common/over-represented in specific population than expected

Question 5

What is the pext score

Answer 5

Proportion expressed across transcripts score: per base expression pattern across transcripts and exons as well as in tissue of interest

Question 6

When is the pext score useful in gnomAD?

Answer 6

Gives biological relevance of variant. When given variant is LoF and strong evidence for disease causing. A low pext score (<0.2) suggests variant not biological relevant (as it’s not expressed across the transcripts or across tissues of interest).

Question 7

What is a Mendelian disease and some examples

Answer 7

Aka monogenic disorders. Caused by mutations in single gene.
Eg. cystic fibrosis, Huntington’s, Sickle Cell, Duchenne’s, Tay-Sach, PKU, Marfan, ADPKD

Question 8

Define Expressivity and Penetrance

Answer 8

Expressivity: Severity of the phenotype that develops in patient with the pathogenic variant
Penetrance: the proportion of individuals carrying the pathogenic variant who display a phenotype

Question 9

What is the “seed sequence” in relation to CRISPR

Answer 9

10-12 bps adjacent to the PAM (3’ end of the gRNA) that determines Cas9 specificity
- 1-5 bps = true seed region (from immunoprecipitation and ChIP-seq data - Zhang 2015)

Question 10

What are the causes of the LOF pathogenic variants appearing in GnomAD?

Answer 10

• Transcript error
• Sequencing error
• Mapping error
• Last exon
• Other annotation error
• Rescue

Question 11

What is homopolymer

Answer 11

Homopolymer refers to a stretch of DNA or RNA sequence where only one type of nucleotide is repeated consecutively, eg AAAAAAAAA

Question 12

What is a “rescue splice variant”

Answer 12

A type of rescue mechanism in which alternative splicing of mRNA mitigates effect of LOF/pathogenic mutation, which preserves function of protein

Question 13

Definition of nonsense-mediated decay

Answer 13

Surveillance pathway that reduces errors in gene expression by eliminating mRNA transcripts that contain premature stop codons

Question 14

Types of mRNA surveillance pathways

Answer 14

1. Nonsense mediated decay (NMD)
2. Nonstop mediated decay (NSD)
3. No-go mediated decay (NGD)

Question 15

How does the location of the termination codon (from truncating mutations) in the last exon affect NMD?

Answer 15

The location of the last exon-exon junction complex (EJC) relative to stop codon NB. If stop codon downstream or within 50 nucleotides of final EJC, transcript translated normally. If upstream >50 nucleotides, NMD occurs.

Question 16

Why does truncating mutations in the last exon generally not pathogenic

Answer 16

• Not subject to NMD
• mutations in 3’ UTR region
• Protein truncation tolerance - critical domains not affected
• haploinsufficiency tolerance - better toeralted if functional copy still present

Question 17

Define linkage disequilibrium

Answer 17

Tendency of alleles to be transmitted more or less often than expected by chance alone - usually caused by close proximity of genes on the same chromosome

Question 18

Define epistasis

Answer 18

Phenomenon in genetics whereby the effect of a gene mutation is dependent on the presence or absence of mutations in one or more other genes, termed modifier genes

Question 19

Define heritability

Answer 19

The measure of proportion of the phenotypic variance of a population that can be attributed to genetic differences

Question 20

What is the “missing heritability” question

Answer 20

• clear conclusion from multiple GWAS studies that highly significant hits accounts for small proportion of the heritability of disease
• amount of heritability explained by GWAS findings much smaller than estimated heritability from family and twin studies

Question 21

Narrow sense heritability (h2)

Answer 21

Narrow-sense heritability (h2) is an important genetic parameter that quantifies the proportion of phenotypic variance in a trait attributable to the additive genetic variation generated by ALL causal variants

Question 22

What are the explanations for the missing heritability problem?

Answer 22

1. large number of common variants of small effects not yet discovered
2. rare variants with large effect sizes not tagged on genotyping arrays
3. overestimation of h2 (narrow sense heritability) in siblings/families due to environmental factors or epigenetics

Question 23

Define tag SNP

Answer 23

A representative SNP in a genomic region with high LD that represent/called a haplotype

Question 24

Define fine-mapping

Answer 24

Process of determining the genetic variant(s), ie causal variant(s), responsible for complex traits, given evidence of association of genomic region with a trait and assuming at least one causal variant exists

Question 25

Types of chromatin annotations

Answer 25

1. Open chromatin regions (indicate regions available for TF binding) 2. histone modifications (highlights enhancer and promotor regions) 3. DNA methylation

Question 26

What are DNAse Hypersensitive Sites (DHS)?

Answer 26

- DHS are regions of DNA that are particularly accessible to cleavage by DNAse I, characterised by lack of nucleosomes. - Indicates regions of high regulatory activity/regulation of gene expression. Correspond to regulatory elements eg promotors, enhancers, silencers etc. - Used in SNP enrichment analysis (a type of chromatin mark)

Question 27

What is ATAC-Seq

Answer 27

Assay for Transposase-Accessible Chromatin with sequencing. Technique used to investigate chromatin accessibility at genome-wide scale. Used to identify areas of open chromatin - ie areas of high regulatory activity (promotor, enhancers and TF binding sites)

Question 28

Steps of ATAC-Seq

Answer 28

1. Transposase tagmentation: uses hyperactive Tn5 transposase enzyme that simultaneously fragments DNA and adds adapters to ends of DNA 2. Selective fragmentation: Tn5 transposase selectively inserts adapters into regions of open chromatin 3. Library preparation: PCR amplification of tagged sequences 4. Sequencing

Question 29

What is pleiotropy

Answer 29

A phenomenon when one gene influences two or more seemingly unrelated phenotypic traits, aka a gene that exhibits multiple phenotypic expression

Question 30

What is allelic heterogeneity

Answer 30

Phenomenon in which different variants (alleles) in the same gene cause the same or similar phenotype

Question 31

Difference between epistasis and allelic heterogeneity (AH)

Answer 31

Epistasis: effect of one gene variant affects (or is dependent of) another gene variant at a DIFFERENT locus AH: different mutations within SAME locus of SAME gene influence the particular trait

Question 32

What are phenocopy conditions?

Answer 32

Variations in phenotype that is caused by environmental conditions and not by genotype

Question 33

Which domains in MYH7 are mostly affected in HCM

Answer 33

Globular head and hinge regions

Question 34

What is the Non-stop Decay pathway and its mechanisms

Answer 34

NSD - targets mRNA (and peptide) for degradation if lacking a proper stop codon (ie the translation keeps going after where the stop codon should've been). 1. Recognition of non-stop mRNAs - ribosome stalls and signals NSD machinery 2. Ski complex (Ski2, Ski3 and Ski8) recruited to stalled ribosome, interacts with exosome (3' to 5' exonuclease activity) 3. Ribosome disassembled (by Pelota-Hbs1) - recycles ribosome 4. Faulty mRNA degraded by exosome assisted by Ski complex 5. Proteosomal degradation after ubiquitination

Question 35

Causes of non-stop mRNAs

Answer 35

Errors in transcription, splicing or premature polyadenylation

Question 36

Causes of ribosome stalling

Answer 36

1. Defective mRNA: - non-stop mRNA - Damaged mRNA - Secondary structures within mRNA (eg hairpins) - Rare codons (due to low availability of corresponding tRNAs) 2. Amino acid deprivation - leading to shortage of charged tRNA 3. Aberrant translation events - misincorporation of amino acids or other errors during translation 4. Protein quality control mechanisms - interaction with faulty nascent polypeptides that do not fold properly

Question 37

Slipped Strand Mispairing

Answer 37

SSM, or Replication Slippage mutation process which occurs during DNA replication. It involves denaturation and displacement of the DNA strands, resulting in mispairing of the complementary bases. Slipped strand mispairing is one explanation for the origin and evolution of repetitive DNA sequences Leads to dinucleotide or trinucleotide repeats At sites of tandem repeats

Question 38

Key mechanisms of ASO action

Answer 38

1: RNA degradation via RNAse H1 - binding of ASO to mRNA = DNA-RNA duplex --> recruits RNase H1 --> cleaves RNA strand = degradation (gapmers) 2. RNA degradation using RNAi - using siRNAs --> recruited into RISC (RNA-induced silencing complex) --> cleaves target mRNA 3: Steric blocking - binding of ASO to mRNA physically blocks access to splicing factors or ribosomes = blocks splicing and/or translation 4. Modulation of splicing (eg exon skipping or exon inclusion): In exon skipping, ASOs designed to target splice sites in pre-mRNA - inhibits binding of spliceosome --> skips over exon (with frameshift mutation eg in Duchenne's) and removes segment from mRNA; in exon inclusion, ASO can sterically block intronic splicing silencer (in SMA)

Question 39

Monocistronic vs polycistronic mRNA

Answer 39

Monocistronic = mRNA translates only 1 single protein chain Polycistronic = multiple ORFs that translate into multiple peptides

Question 40

Examples of gene expression roles of UTRs (untranslated regions) of mRNAs

Answer 40

mRNA stability, mRNA localisation and translational efficiency

Question 41

What are nuclear speckles

Answer 41

Regions in the nucleus associated with pre-mRNA splicing and transcriptional regulation Aka interchromatin granule clusters

Question 42

What is MALAT-1

Answer 42

MALAT-1 = metastasis-associated lung adenocarcinoma transcript 1 - long non-coding RNA lncRNA widely expressed in many tissues with roles in gene expression and splicing etc - localised in nuclear speckles - over-expressed in many CAs (eg lung, breast, liver) - promotes proliferation

Question 43

Key differences between gapmers and mixmers

Answer 43

2 different types of ASOs Gapmers = central DNA gap flanked by modified nucleotides vs Mixmers = alternating modified nucleotide Gapmer = RNase-H mediated RNA degradation vs Mixmer = steric blocking Gapmer = long-lasting vs mixmer = transient

Question 44

What is an operon

Answer 44

Functioning unit of DNA containing a cluster of genes under the control of a single promoter. Genes transcribed together into an mRNA and translated together or spliced into monocitronic mRNAs Common in prokaryotes and rare in eukaryotes

Question 45

Structure of an operon

Answer 45

1. Promoter - nucleotide sequence that enables a gene to be transcribed 2. Operator - DNA segment to which a repressor binds, defined in the lac operon as between the promoter and structural genes 3. Structural genes Others: - repressor protein (coded by regulatory gene) - inducer - displaces repressor

Question 46

Explain the lac operon mechanism

Answer 46

Encoded in E. coli Mechanism in which the bacteria switches on the transcription of enzymes that processes lactose when glucose is low. Not always fully active as waste of energy. Always background expression (for lac Y - B-galactoside permease) to enable detection of lactose in cell Lac operon: 3 structural genes, promoter, terminator, regulator and operator lacZ = B galactosidase (cleaves lactose into glucose and galactose) lacY = B-galactoside permease - transmembrane symporter pumps B-galactosides into cell lacA = B-galactoside transacetylase - transfers acetyl group from acetyl-CoA to thiogalactoside In absence of lactose, repressor is bound to the operator, repressing transcription (by blocking DNA dependent RNA polymerase), albeit imperfectly = background expression Presence of lactose (but not glucose), binds to repressor and inactivates it = transcription

Question 47

Types of RNA secondary structures

Answer 47

Helices Hairpin loop Bulge loop Interior loop Junction/Intersection

Question 48

What is the cre-lox recombination

Answer 48

Gene editing (deletion/inversion and translocation) technique that allows for spatiotemporal control. Derived from P1 bacteriophage. Cre Recombinase = 38kDa enzyme that recognises Lox sites - catalyses recombination between them: - if LoxP same direction = deletion; opposite directions = inversion; interchromosomal recombination = translocation Used in lineage tracing as reporter gene (GFP) can be activated by Cre to track cell populations (using cell-specific promoters)

Question 49

What is the Bayesian Pairwise Analysis

Answer 49

Statistical approach used to model pairwise relationships between 2 variables, incorporating PRIOR KNOWLEDGE or ASSUMPTIONS about the data distribution. Involves updating PRIOR BELIEFS with new evidence to form a POSTERIOR DISTRIBUTION

Question 50

Concept of Locus (Loci), Alleles, LD, Tag SNP and Haplotype NB!!!

Answer 50

A LOCUS is a specific location on the chromosome. Depending on context, it refers to either a specific base-pair position for Single-Base Pair Locus (eg rs-113488022 at chr14:23822594), or Multi-Base Pair Locus (referring to an entire gene - gene locus, structural variants - SV locus, QTLs - eg eQTL locus, or GWAS-locus). The ALLELE is the version of the gene at a specific LOCUS, and also depends on context: for Single-Base Pair Locus, it is the specific SNP (A,T,C or G) at that location, or for Multi-Base Pair locus, it refers to the entire SEQUENCE of nucleotides. LD - Linkage Disequilibrium - refers to the NON-RANDOM association of ALLELEs at different LOCI in a population, where certain ALLELES (eg SNPs) are inherited more often than expected by chance due to physical proximity on a chromosome (due to CROSSING OVER during meiotic recombination). The TAG-SNP is a representative SNP in a region of genome with high LD that represents a group of SNPs called a HAPLOTYPE. Therefore a Haplotype is a group of SNP in high LD with each other.

Question 51

Concepts of fine mapping, SNP enrichment and Colocalisation studies

Answer 51

Fine-mapping: methods aimed to define causal VARIANTS SNP-enrichment: prioritise disease-relevant CELL TYPES Colocalisation: nominates likely target GENES