Genome, Epigenome and Inheritance Flashcards
What is the structure of DNA
Double stranded helix from two anti-parallel strands
Phosphodiester backbone
Pentose sugar - purines (A and G) and pyramidine (T and C)
What is the structure of chromatin
Nucleosomes - Histone octamer’s
2A, 2B,3 and 4
H1 binds with linker DNA (DNA between nucleosomes)
The nucleotides and H1 stack to form solenoids
Euchromatin and heterochromatin form depending on how tightly bound they are (loose and tight respectively)
Also related: trithorax and polycomb proteins which impact euchromatin and heterchromatin formation
What are the functions of centromeres
They keep the sister chromatids together
They attach to microtubules doing cell division
They are rich in heterochromatin - and are normally highly repetitive sequence of CAG
What are the functions of telomeres
They have a specific six base repeated sequence that protects chromosomes from being degraded TTAGGG
They are repaired by telomerase but this is only active in certain cell types - inappropriate activation can lead to cancer
Describe and explain the functional units of a gene
Exons - codes for amino acids (except the 5’ and 3’ UTR)
UTR - contains regulatory elements important for the control of protein synthesis
Introns - non-coding sections of genes between exons
Promoter - 5’ of gene containing important regulatory elements for transcription some genes include transcription factors which combined promoters and other motifs
Enhancer - TF binding site to enhance RNA pol recruitment
Silencer - TF binding site to inhibit RNA pol recruitment
Define the genome
The genome is the entire set of DNA/chromosomes in the human body nuclear and mitochondrial
Define the exome
The set of genes which have coding functions
Define the epigenome
The chemical changes to the DNA and histone proteins which can be passed on to offspring
It alters chromatin structure recruits histone modifiers, represses transcription, enables differential gene expression
It is established as a genome wide pattern at fertilisation
It responds to environmental cues cellular and extracellular
Non-mendelian
What is differential gene expression
The processes that determine which genes are actively transcribed and translated into mRNA and proteins in a cell and under what conditions
In time - temporal
Development i.e. embryo versus adult
In response to hormones, infection and other signals
Spatially
Different tissues/cells expressed different genes e.g. brain versus liver
Overview of transcription
Transcription factors find the DNA and promote or repress transcription
RNA polymerase unwinds dsDNA separating the sense and antisense strand. It then recruits nucleotides to the antisense strand.
Sense strand = contains the same sequence as mRNA (5’ to 3’)
Antisense strand = template to generate mRNA (3’ to 5’)
Modifications of mRNA then follow…
Describe the modifications of mRNA
Capping - adding of altered/methylated guanosine
Protects 5’ from extension and degradation stabilises the molecule
Facilitates transport into the cytoplasm
Enhances translation
Polyadenylation - adding of 50-250 I adenosines by polyadenylation polymerase
Protects the 3’ end from degradation
Splicing - removal of introns and joining together of exons via the spliceosome, which cleaves the 5’ site which loops onto 3’ site , following which the in the loop is cleaved off
This is directed by sequences at the exon-intron boundaries and those within the intron
Alternative/differential slicing helps to develop a different isoforms of protein using different tissues and/or different stages of development
Overview of translation
Translation occurs in the cytoplasm, facilitated by ribosomes (rRNA) and tRNA
Not all components are translated, such as the polyA tail, and the UTR regions
There are important sites in the UTR which give signals in translation Start codon (AUG) and stop codon (UAA/UAG/UGA)
Define synonymous mutations
These are also known as silent mutations, where the base change does not result in an amino acid change
Define non-synonymous mutations (missense and nonsense)
Missense - amino acid substitution
Depends on
Physiochemical similarity between the two amino acid
Functional role of the specific domain of the protein
Phylogenetic conservation of original amino acid amongst diver species
Nonsense - stop codon
If it appears any on it may be subject to nonsense mediated decay
However, there could be a truncated mRNA/protein - impact depends on where it occurs
Define indel mutations (frame shift and non-frame shift)
These are insertions or deletions
This includes unequal crossover during meiosis, resulting in loss in one chromosome, and gain in another - or due to polymerase slippage.
Generally in-frame mutations do not cause a huge problem
Some diseases such as expansion disorders can cause a disease phenotype
What are the effects of variance in non-coding regions
Promoter region variants – affects gene expression
Terminator sequence variance – affect the correct termination and polyadenylation of mRNA
Spicing variance – lead to creation or deletion of the spice donor/acceptor or branch site
This can lead to incorrect incorporation of introns, or exon skipping
It can be exonic or intronic
What is loss/gain of function and dominant negative
Loss of function
Reduced activity/decreased stability – hypomorph
Complete loss of gene product – null allele/amorph
Gain of function
Increases levels of gene expression and/or new function for protein products
Dominant negative
Mutant allele produces gene product that interferes with the correct role
Examples of loss of function diseases
Recessive
Sickle-cell anaemia, phenylketonuria, cystic fibrosis, gauchers disease, haemochromatosis
Dominant (haploinsufficiency)
MonoMac syndrome 1 lack of GATA2 = monocytes and B cell deficiencies
CHARGE syndrome, Marfan syndrome, Ehlers-Danlos syndrome
Examples of gain of function diseases
Dominant Achondroplasia – gain of function mutation in FGFR3 leads to decrease bone mass by altered regulation of osteoblast/class activity
What are dominant negative diseases
Mutations in transcription factors removes activation domain but still binds DNA = can’t trigger transcription
Mutation in proteins that function as a dimer, but may lack functional domains = can dimerise with WT but the dimer is non-functional
Can occur in sodium channels
What happens when you fail to regulate gene expression
Metabolic disease
Cellshape/motility – metastasis
Cell differentiation – congenital disorders
Cell proliferation – cancer
What is the main mechanism of gene expression regulation
It is mainly regulated at the level of transcription it can be transcribed to different levels
Abundant such as housekeeping genes EG glycolytic enzymes
Rare
None – tissue-specific e.g. globin, non-existent in some highly transcribed in others
State some gene control elements
Transcription factor binding regions including -
Promoter
Regulatory elements
Enhancers
Silences
(Specificity is derived from specific transcription factors which only target a specific gene or family of genes)
Post-translational gene regulation
Small non-coding RNA
Describe control of gene expression through promoters
Promoters have recognition sequences responsible for recruiting RNA polymerase and transcription factors
A key sequence is the TATA box - this recruits the general transcription factor TATA box binding factor
Transcriptional activators recruit RNA polymerase
Transcriptional represses prevent transcription by RNA polymerase
Describe control of gene expression through regulatory elements
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)
Describe control of gene expression through enhancers
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
Describe the control of gene expression through silencers
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
Describe post-transcriptional gene regulation
Polyadenylation
Capping
Splicing
Translation - 5’ UTR determines how efficiently the ribosome initiates translation
RNA stability - conferred by the 3’ UTR, and impacted by miRNA
Describe Beta Thalassaemia as an example of disease caused by a fault in control of gene expression
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
Function of Trithorax and Polycomb proteins
Trithroax - maintain expression
Polycomb - prevent expression
What is the difference between general and specific transcription factors
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
What is the significance of alternative splicing
Alternative/differential slicing helps to develop a different isoforms of protein using different tissues and/or different stages of development
What is the structure and function of miRNA’s
Structure - ssRNA
Function - post-transcriptional regulation
Binds to complementary sequences
Strong binding = rapid degradation
Weaker binding = some degradation
Occurs in cytoplasm
Definite epigenetics
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
Examples of epigenetic phenomena
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
How are induced pluripotent cells and nuclear reprogramming evidence of epigenetic phenomena
4 TF’s can be used to remove the epigenetic mechanisms from a somatic cell that cause cell differentiation thus leading pluripotency
List 3 epigenetic mechanisms
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
Describe mechanisms of epigenetic regulation
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
Describe nucleosome modifications
Methylation Acetylation Phosphorylation Ubiquitination Other
Typically end terminal tail modifications
Different combinations = different effects
Describe writers, erasers and readers
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
Describe the involvement of CpG dinucleotides
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
How do we detect epigenetic modification (epigenomics) with ChIP-seq NGS
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
How is epigenetics involved in X-inactivation
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
Describe involvement of epigenetics in CHARGE syndrome
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
Describe involvement of epigenetics in cancer
DNA hypermethylation = oncogenic mechanism
Histone deacetylase = switch tumour suppressor genes off
*HDAC Inhibitors = tested for cancer treatment to reactive TS genes
What factors influence DNA methylation
Aging
Diet - intake of methyl groups via folate
Environment -
Arsenic exposure = hypomethylation of RAS
Cadmium exposure = inactivation of DNMT1 thus global hypomethylation
Link methylation and anxiety
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
Describe the mendelian modes of inheritance
AR - homozygote V compound heterozygote (trans/cis)
AD
X-linked recessive/dominant
Y-linked
What is variable expressivity
Variation severity/symptoms of disorder between individuals with same mutn
What is incomplete/reduced penetrance
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
What is sex-influences/limited inheritance
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
What is pleiotropy
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
What is mosaicism
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
What is phenocopy, genocopy, heterogeneity
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
Describe X-linked Recessive inheritance
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)
What is anticipation
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
What is uniparental disomy
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
Describe UPD disorders
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
Describe the complexities of mitochondrial inheritance
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
Explain and give examples for monogenic, digeneic, polygenic and multifactorial disorders
Monogenic - 1 gene -cystic fibrosis, DMD
Digenic - 2 genes - ?
Polygenic - multiple genes, often multifactorial
Multifactorial - polygenic + environmental factors - schizophrenia
What is a quantitative trait
A measurable phenotype that depends on the cumulative actions of many genes and the environment
Continuous V discontinuous
What is susceptibility
Environment needed to trigger disease phenotype, threshold effect
Most multifactorial disorders have a threshold
Discontinuous traits follow distribution but phenotype is determined by threshold
What methods are used to investigate monogenic disease
Whole exome sequencing
Linkage analysis
Auto-zygosity mapping
What methods are used to investigate multifactorial disease
GWAS
Case-control
What is an imprinted gene
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
Explain how chromosome rearrangement can lead to imprinting disorders
If a gene is deleted/duplicated - then there will be in imbalance between paternal and maternal patterns
One will be lost
Explain how aberrant methylation patterns (epimutation) can lead to imprinting disorders
Imbalance in methylation patterns can mean overactivation or underactivation
Explain how uniparental disomy can lead to imprinting disorders
There may be double paternal or double maternal patterns = imbalance
What is the conflict hypothesis
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
Describe Prader-Willi syndrome
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
Describe Angelman syndrome
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
What and how can we detect PW deletions
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
What is the transcriptome
Complete set of transcripts (RNA) expressed in a sample/tissue at specific point of time
Tissue specificity
Changes in response to stimuli/disease
What post-transcriptional modifications enhance complexity in our genes
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
What does alternative splicing achieve
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
Describe splicing mechanisms
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
Describe aberrant splicing
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
Describe splicing mutations
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
Describe the splicing mutation disease within the LMNA gene
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)
Describe the methods for functional testing of predicted splice variants
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
Describe the use of RT-PCR in beta thalassemia and leigh syndrome
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
Describe when the minigene assay is use to analyse RNA
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
Discuss why WES and WGS is not ideal in analysing RNA
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
Describe transcriptome analysis
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
Describe RNA sequencing library prep
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
Describe the use of RNAseq in routine diagnostics
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
