Genome, Epigenome and Inheritance Flashcards

Question

Describe control of gene expression through regulatory elements

Answer 1

These can act as transcription factors to allow recruitment of general transcription factors and RNA polymerase to the TATA box Example the oestrogen response element This binds to the oestrogen receptor and forms a complex that acts as a transcription factor allowing recruitment to be TATA box (Explored further in mice models)

Answer 2

These are sequences of DNA that act to enhance the recruitment of RNA polymerase to a promoter They contain DNA sequences that are strong binding site for transcription factors

Answer 3

These are sequences of DNA that are adjacent to transcription, acting to inhibit RNA polymerase (5’, 3’ or intronic) They may be able to mask the activity of an enhancer Direct interaction with a general transcription factors Bind sites to prevent RNA polymerase being recruited by them and other transcription factors binding

Answer 4

Polyadenylation Capping Splicing Translation - 5’ UTR determines how efficiently the ribosome initiates translation RNA stability - conferred by the 3’ UTR, and impacted by miRNA

Answer 5

A group of genetic diseases caused by insufficient expression of β-globin Most types of beta thalassaemia the protein is structurally normal There are multiple forms of the disease Causal mutations TATA box point mutation = failure to recruit RNA polymerase Splice site point mutation = truncated mRNA

Answer 6

Trithroax - maintain expression Polycomb - prevent expression

Answer 7

General - bind generally to promoters to then enhance a cascade of TF activation Specific - recruit RNA polymerase to genes that need to be transcribed, targeting only a specific gene or gene family thus deriving specificity

Answer 8

Alternative/differential slicing helps to develop a different isoforms of protein using different tissues and/or different stages of development

Answer 9

Structure - ssRNA Function - post-transcriptional regulation Binds to complementary sequences Strong binding = rapid degradation Weaker binding = some degradation Occurs in cytoplasm

Answer 10

The study of heritable changes in gene expression not due to changes in the DNA sequence Heritable can be defined on the cellular or organism level i.e. changes inherited by subsequent generations of cells or organisms

Answer 11

Cell differentiation and memory - includes trithorax and polycomb proteins to drive expression to maintain cellular identity Genomic imprinting - e.g. Rainbow and copycat, where the clone looked different due to the removal of imprinting Development plasticity and the environment - polyphenism (distinct phenotypes elicited by environment e.g. sex determination in clown fish or temperature changing alligator) Transgenerational epigenetics Diseases with epigenetic basis or contribution

Answer 12

4 TF's can be used to remove the epigenetic mechanisms from a somatic cell that cause cell differentiation thus leading pluripotency

Answer 13

These mechanisms interact with TF's to regulate gene-expression patterns inherited from cell to cell DNA methylation Post-translational modification of histone proteins Small and non-coding RNA The patterns underlie embryonic development, differentiation and cell identity

Answer 14

Regulatory elements - influencing gene transcription Altering chromatin structure Trithorax protein = heterochromatin into euchromatin (H3K4me3) Polycomb protein = euchromatin into heterochromatin (H3K27me3) Chromatin remodelling is important in establishing and maintaining cellular identity

Answer 15

``` Methylation Acetylation Phosphorylation Ubiquitination Other ``` Typically end terminal tail modifications Different combinations = different effects

Answer 16

Writers - enzymes that add epigenetic marks to histones or DNA E.g. histone methyltransferase or DNA methyltransferase Erasers - enzymes that remove these marks E.g. histone demethylase Readers - proteins/protein domains that can recognise these marks through which they are recruited to DNA and introduce further modifications E.g. remodel chromatin, recruit other writers or erasers These are critical for normal development and mutations result in disease A singular protein may have multiple functions, e.g. have a reader domain, and writing activity

Answer 17

Only cytosine residues are methylated during DNA methylation Cytosines found in CpG pairs tend to be most methylated Methyl CpG recruits specific readers e.g. methyl-binding proteins (MBP/MeCP) These protein recruit additional enzymatic proteins complexes that compact DNA - heterochromatin formation and gene repression *DNA methylation does not always lead to heterochromatin, but it is the most common effect

Answer 18

ChIP-seq (chromatin immunoprecipitation) Proteins are crosslinked to DNA, isolated and then sonicated into fragments They are then incubated with antibodies for a specific histone modification e.g. H3Ly27me3 The Ab/chromatin complexes are isolated, and then the Ab removed DNA is then used in NGS

Answer 19

X-inactivation is regulated by the locus Xic Xic encodes the ncRNA module Xist Xist coats the X chromosome in cis, and recruits modifying enzymes like polycomb = heterochromatin, compaction (Barr body) and gene repression

Answer 20

AD condition affecting multiple body systems, 60-90% of cases caused by CHD7 mutation CHD7 is an ATP-dependent chromatin remodelling factor - moves nucleosomes around/removes histones

Answer 21

DNA hypermethylation = oncogenic mechanism Histone deacetylase = switch tumour suppressor genes off *HDAC Inhibitors = tested for cancer treatment to reactive TS genes

Answer 22

Aging Diet - intake of methyl groups via folate Environment - Arsenic exposure = hypomethylation of RAS Cadmium exposure = inactivation of DNMT1 thus global hypomethylation

Answer 23

Rat study - methylation in the brain and anxiety Low licking grooming of offspring - increased methylation, less resilient to anxiety High licking/grooming of offspring - decreased methylation , more resilient to anxiety Monozygotic twins study Significant discordance in several diseases e.g. cancer, schizophrenia

Answer 24

AR - homozygote V compound heterozygote (trans/cis) AD X-linked recessive/dominant Y-linked

Answer 25

Variation severity/symptoms of disorder between individuals with same mutn

Answer 26

Percentage of individuals who carry the mutation V develop symptoms of the disorder Influenced by modifier genes, environment, lifestyle, other non-genetic biological factors (e.g. hormones) Many dominant disorders show age-dependant penetrance E.g. cancers, Huntington's disease, polycystic kidney disease

Answer 27

Over-representation of condition in one sex due to other biological factors related to sex e.g. hormones E.g. homozygous females with AR hemochromatosis much less likely than males to show symptoms because of 'rescue effect' of menstruation

Answer 28

Mutn in one gene affects multiple organs/systems or leading to different presentations There may be no apparent relationship between the various symptoms Often seen when the gene is involved in early development

Answer 29

Somatic - some normal and some abnormal, doesn't affect sperm/eggs Arises in early embryogenesis, only in some tissues/cells Germ-line - some normal and some abnormal cell, affects sperm/eggs Mutation present in variable proportion of gametes - easy to test from sperm Children are not mosaics for that mutation Can infer this from pedigree if multiple children get a seemingly 'de novo' mosaicism

Answer 30

Phenocopy - same phenotype arising due to non-genetic reasons Genocopy/Heterogeneity- same disorder arises due to mutn in different genetic loci Locus heterogeneity- when the same disorder can be caused by mutations in different genes .e.g. AR Retinitis pigmentation Allelic/mutational heterogeneity - when different mutations in the same gene cause one condition e.g. cystic fibrosis

Answer 31

X-linked genes never passed from father to son - ALL daughters of affected males are obligate carriers Children of carrier females have a 50% chance of inheriting the mutant allele Skewed X inactivation means that some woman will still manifest symptoms in cells where the healthy X is inactivated (manifesting carrier)

Answer 32

Disease occurs earlier and/or with greater severity in subsequent generation Typically occurs in repeat expansion disorders e.g.. myotonic dystrophy Harmful protein gets bigger and may even impede transcription of other proteins due to its size There is a tendency for repeat section to increase further due to slippage

Answer 33

Occurs when an individual has inherited both homologous chromosomes from a single parent - due to non-disjunction - including trisomic rescue NDJ Meiosis I = heterodisomy = 2 different chromosomes from one parent NDJ Meiosis II = isodisomy = 2 same chromosomes from one parent Trismic rescue When parent 2 is involved, the cell gets 3 chromosomes, but parent 2's chromosome is kicked out

Answer 34

X-linked disorders can be passed from affected father to son Occurs if NDJ happens in meiosis I, leading to XY in one chromosome - upon fertilisation, this becomes XXY. This could revert back to fathers XY by trismic rescue Child of a couple can have an autosomal recessive disorder, despite only one parent being a carrier It can result in imprinting disorders, such as Prader-willi or Angelman syndrome

Answer 35

Maternal inheritance only Every cell has many different mitochondria If all are the same = homoplasmy If there is a mixture = heteroplasmy, can vary in percentage Higher percentage = greater likelihood of disease manifestation IMPORTANT - here are mitochondrial disease that are due to mutation in nuclear DNA, can show mendelian inheritance

Answer 36

Monogenic - 1 gene -cystic fibrosis, DMD Digenic - 2 genes - ? Polygenic - multiple genes, often multifactorial Multifactorial - polygenic + environmental factors - schizophrenia

Answer 37

A measurable phenotype that depends on the cumulative actions of many genes and the environment Continuous V discontinuous

Answer 38

Environment needed to trigger disease phenotype, threshold effect Most multifactorial disorders have a threshold Discontinuous traits follow distribution but phenotype is determined by threshold

Answer 39

Whole exome sequencing Linkage analysis Auto-zygosity mapping

Answer 40

GWAS Case-control

Answer 41

The process by which one parental allele is preferentially silence according to its parental origin More than half of the imprinted genes are involved in pre and post-natal growth There are imprinting control regions which regulate pattern of expression e.g. methylation

Answer 42

If a gene is deleted/duplicated - then there will be in imbalance between paternal and maternal patterns One will be lost

Answer 43

Imbalance in methylation patterns can mean overactivation or underactivation

Answer 44

There may be double paternal or double maternal patterns = imbalance

Answer 45

Paternally expressed genes promote growth, maternal expressed genes supress growth During gametogenesis, the epigenetic markings are erased, and re-established It is established according to gender - maternal epigenome is applied to oocytes, paternal to sperm

Answer 46

Defect in chr15q11.2 - increased maternal gene effect Deletions - no paternal genes (only suppression) - 75% Maternal UPD - both maternal genes (only suppression) - 25% Epigenetic defect - paternal IC hypermethylated (only suppression) - 1% ``` Clinical features Moderate to severe learning difficulties, average IQ = 60 Better at visual-spatial problems 80% have behavioural problems Hypogonadotropic hypogonadism ```

Answer 47

Defect in chr15q11.2 - increased paternal gene effect Deletions - no maternal gene (no suppression) - 70% Paternal UPD - two paternal genes (no suppression) - 2-5% Epigenetic defects - maternal IC hypomethylated (no suppressors) - 2-5% Mutations in UBE3A so it's not expressed/non-functional - 20% Clinical features in all patients: Severe cognitive impairment, receptive and non-verbal skills better than verbal, ataxia or tremulousness of gait Behavioural uniqueness - frequent laughter/smiling, happy disposition, short attention span, easily excitable Other clinical features - microcephaly, seizures, sleep disturbance

Answer 48

PML probe binds both Chr.15 at the critical region and another region Normally it binds SNRPN (critical region) and PML (normal) Control - 2 PML detected Deletion = only 1 SNRPN

Answer 49

Complete set of transcripts (RNA) expressed in a sample/tissue at specific point of time Tissue specificity Changes in response to stimuli/disease

Answer 50

Enhancing complexity encoded in our genes: Variation in genome, epigenome, proteolytic cleavage of protein, phosphorylation, acetylation Proteome and metabolome - quantitative mass spectrometry is used for analysis Genomics, transcriptomics, epigenomics - next generation sequencing is used for analysis

Answer 51

Alternative splicing >90% of human genes - tissue specific/developmental variants Inclusion or exclusions of exons Increased/decreased UTR Intron retention - used to increase or decrease expression of a protein

Answer 52

Recognition of splice sites > intron removal Components - several cis and trans acting elements Spliceosome complex (trans-acting element) - Work with the cis elements below and other splicing repressors/activators Donor and acceptor sites - these are evolutionarily conserved For 98.7% of splice sites - 5' Donor = GT (GU), 3' Acceptor = AG (canonical pair) - Most frequent non-canonical pair = GC/AG (0.56%) and AT/AC (0.09%), Donor site mutation more prevalent than acceptor 1.5:1 Branch point, polypyrimidine tract - Highly degenerated and are recognised with the donor and acceptor sites by spliceosome Enhancer and silencer splicing sequences

Answer 53

Aberrant splicing (9% of all mutations) Splice site mutations Splicing factor-binding mutations Intronic variants Closer to donor/acceptor splice regions Deep intronic variants - within middle of introns Synonymous variants Exonic - usually these type of variants are investigated by looking at the protein itself, not splicing and are usually ignored as they usually don't affect the protein Need to test splicing changes via testing the RNA in a patient

Answer 54

Canonical splice site splicing mutation - whole exon skipping Canonical splice site variants - usage of cryptic/pseudo or intronic splice site thus inclusion of intron, or exon fragment skipping Deep intronic variants - inclusion of cryptic/pseudoexons SNV in exons - create new splice site, thus losing an exon fragment Splicing mutations in Disease: Different splice mutations in LMNA cause distinct disease Exonic splice enhancer mutation - exon skipping

Answer 55

Different splice mutations within the LMNA gene causes different distinct diseases LMNA proteins are found in the nucleus and important in maintaining nuclear shape Mutations Limb girdle muscular dystrophy 18 (LGMD18) - mutant 5' ss (c.1608+5G>C) Retention of intron 9 = premature stop codon Familial partial lipodystrophy type 2 (FPLD2) - mutant 5'ss (c.1488+5G>C) Retention of intron 8 = 8 premature stop codon Hutchinson-Gilford progeria syndrome - alternative 5'ss (c.1824C>T) Within exon = activates cryptic ss = exon 11 150bp deletion ``` Dilated cardiomyopathy (DCM) - alternative 3'ss (c.640-10A>G) Exon 4 5' extension = protein +3AA (non-frameshift) ```

Answer 56

Reverse-transcriptase PCR (RT-PCR) RNA from patient fibroblasts or PBMCs (or other biopsied tissue) Oligo-DT (random primers) anneal to RNA Specifically used as they anneal to the poly-A tail of mRNA Reverse transcriptase forms cDNA, followed by PCR Problem of nonsense mediated decay (NMD) Can be inhibited by treating cells with puromycin Minigene assay Cells or tissues not available Introduce variant into healthy cells using genome-editing (e.g CRISPR-Cas9). Test with RT-PCR

Answer 57

Beta Thalassemia >200 known mutations in HBB including splice mutations Example - deep intronic variant results in intronic retention RT-PCR enables visualisation of the larger fragment via electrophoresis Leigh Syndrome - MRPS34 Donor mutation activated a cryptic ss leading to partial deletion and frame shift mutation One acceptor site mutation lead to 2 different aberrant transcripts These transcripts are distributed differently in tissues This makes validation of splice site mutations as they are tissue specific and may not be detected in all tissues SS mutations aren't 100% effective as they’ll be heterogenous in different tissues/not be included in some

Answer 58

Used when there aren't any cells or tissue samples available to extract RNA Requires cloning of DNA fragment into expression vector This is introduced into cell cultures Expressed transcript is then analysed

Answer 59

Yield - 25-75% depending on cohort Splice variants underrepresented Deep intronic variants not detected in whole-exome sequencing Detecting by genome-sequencing by prioritisation/prediction is difficult Large number of variants across the species, it is difficult to determine what is pathogenic Only donor and acceptor site mutations simple to predict, as they're highly conserved Solution = directly interrogate the transcriptome

Answer 60

RNA-sequencing via NGS (RNA-Seq) allows the entire transcriptome to be analysed in a single run Investigate - specific cell, tissue, or organism at a given developmental stage or physiological condition Long reads of protein-coding mRNA and non-coding RNA such as rRNA, tRNA, small non-coding RNA and large-intergenic non-coding RNA Used to identify genetic variants and their effects Altered expression levels Aberrant splicing Gene fusions

Answer 61

Purify RNA e.g. mRNA = polyA selection > fragmentation > random priming Reverse transcribe into cDNA > ENDREPAIR, phosphorylation, A-tailing > adaptors ligated > PCR amplified > sequenced on illumina

Answer 62

It is in the early stages but used in Cancer – gene fusions Inherited diseases e.g. Neurofibromatosis 1 (NF1) regulatory splicing RNA-seq in Mendelian disorder AR condition where patients already has WES/WGS but molecular diagnosis not made Muscles and skin biopsies were not originally taken, which is why they didn't get tested RNA-seq allowed the molecular diagnosis to diagnose those who were missed in WGS/WES RNA-seq in mitochondrial disorders Fibroblast used, all patients screened with WGS Detected 3 different types of abnormalities depending on what mutation was present ``` Aberrant expression (typically low expression) Mutation likely causes NMD due to frameshift/premature stop ``` Aberrant splicing Inclusion of cryptic exons, exon skipping, exon truncation, exon extension, intron retention Mono-allelic expression Genetic Splicing/premature stop codons leading to NMD thus only the other allele works The other allele could have a missense, thus only faulty protein expressed Promoter or regulatory mutations Large deletions e.g. TAR syndrome Epigenetic mechanisms Therapy Antisense oligonucleotide (AON) therapy can be used to treat splicing diseases It can block sections of DNA to induce splicing/skipping