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Human Medical Genetics > Genome, Epigenome and Inheritance > Flashcards

Genome, Epigenome and Inheritance Flashcards

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USMLE® Step 1 flashcards Biology 101 flashcards
1
Q

What is the structure of DNA

A

Double stranded helix from two anti-parallel strands
Phosphodiester backbone
Pentose sugar - purines (A and G) and pyramidine (T and C)

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2
Q

What is the structure of chromatin

A

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

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3
Q

What are the functions of centromeres

A

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

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4
Q

What are the functions of telomeres

A

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

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5
Q

Describe and explain the functional units of a gene

A

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

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6
Q

Define the genome

A

The genome is the entire set of DNA/chromosomes in the human body nuclear and mitochondrial

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7
Q

Define the exome

A

The set of genes which have coding functions

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8
Q

Define the epigenome

A

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

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9
Q

What is differential gene expression

A

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

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10
Q

Overview of transcription

A

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…

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11
Q

Describe the modifications of mRNA

A

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

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12
Q

Overview of translation

A

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)
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13
Q

Define synonymous mutations

A

These are also known as silent mutations, where the base change does not result in an amino acid change

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14
Q

Define non-synonymous mutations (missense and nonsense)

A

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

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15
Q

Define indel mutations (frame shift and non-frame shift)

A

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

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16
Q

What are the effects of variance in non-coding regions

A

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

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17
Q

What is loss/gain of function and dominant negative

A

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

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18
Q

Examples of loss of function diseases

A

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

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19
Q

Examples of gain of function diseases

A 
Dominant
Achondroplasia – gain of function mutation in FGFR3 leads to decrease bone mass by altered regulation of osteoblast/class activity
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20
Q

What are dominant negative diseases

A

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

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21
Q

What happens when you fail to regulate gene expression

A

Metabolic disease
Cellshape/motility – metastasis
Cell differentiation – congenital disorders
Cell proliferation – cancer

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22
Q

What is the main mechanism of gene expression regulation

A

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

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23
Q

State some gene control elements

A

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

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24
Q

Describe control of gene expression through promoters

A

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

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25
Q

Describe control of gene expression through regulatory elements

A

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)

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26
Q

Describe control of gene expression through enhancers

A

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

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27
Q

Describe the control of gene expression through silencers

A

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

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28
Q

Describe post-transcriptional gene regulation

A

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

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29
Q

Describe Beta Thalassaemia as an example of disease caused by a fault in control of gene expression

A

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

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30
Q

Function of Trithorax and Polycomb proteins

A

Trithroax - maintain expression

Polycomb - prevent expression

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31
Q

What is the difference between general and specific transcription factors

A

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

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32
Q

What is the significance of alternative splicing

A

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

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33
Q

What is the structure and function of miRNA’s

A

Structure - ssRNA

Function - post-transcriptional regulation

Binds to complementary sequences
Strong binding = rapid degradation
Weaker binding = some degradation

Occurs in cytoplasm

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34
Q

Definite epigenetics

A

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

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35
Q

Examples of epigenetic phenomena

A

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

36
Q

How are induced pluripotent cells and nuclear reprogramming evidence of epigenetic phenomena

A

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

37
Q

List 3 epigenetic mechanisms

A

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

38
Q

Describe mechanisms of epigenetic regulation

A

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

39
Q

Describe nucleosome modifications

A 
Methylation
Acetylation
Phosphorylation
Ubiquitination
Other

Typically end terminal tail modifications

Different combinations = different effects

40
Q

Describe writers, erasers and readers

A

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

41
Q

Describe the involvement of CpG dinucleotides

A

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

42
Q

How do we detect epigenetic modification (epigenomics) with ChIP-seq NGS

A

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

43
Q

How is epigenetics involved in X-inactivation

A

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

44
Q

Describe involvement of epigenetics in CHARGE syndrome

A

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

45
Q

Describe involvement of epigenetics in cancer

A

DNA hypermethylation = oncogenic mechanism

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

46
Q

What factors influence DNA methylation

A

Aging

Diet - intake of methyl groups via folate

Environment -
Arsenic exposure = hypomethylation of RAS
Cadmium exposure = inactivation of DNMT1 thus global hypomethylation

47
Q

Link methylation and anxiety

A

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

48
Q

Describe the mendelian modes of inheritance

A

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

49
Q

What is variable expressivity

A

Variation severity/symptoms of disorder between individuals with same mutn

50
Q

What is incomplete/reduced penetrance

A

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

51
Q

What is sex-influences/limited inheritance

A

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

52
Q

What is pleiotropy

A

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

53
Q

What is mosaicism

A

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

54
Q

What is phenocopy, genocopy, heterogeneity

A

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

55
Q

Describe X-linked Recessive inheritance

A

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)

56
Q

What is anticipation

A

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

57
Q

What is uniparental disomy

A

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

58
Q

Describe UPD disorders

A

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

59
Q

Describe the complexities of mitochondrial inheritance

A

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

60
Q

Explain and give examples for monogenic, digeneic, polygenic and multifactorial disorders

A

Monogenic - 1 gene -cystic fibrosis, DMD

Digenic - 2 genes - ?

Polygenic - multiple genes, often multifactorial

Multifactorial - polygenic + environmental factors - schizophrenia

61
Q

What is a quantitative trait

A

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

Continuous V discontinuous

62
Q

What is susceptibility

A

Environment needed to trigger disease phenotype, threshold effect
Most multifactorial disorders have a threshold

Discontinuous traits follow distribution but phenotype is determined by threshold

63
Q

What methods are used to investigate monogenic disease

A

Whole exome sequencing

Linkage analysis

Auto-zygosity mapping

64
Q

What methods are used to investigate multifactorial disease

A

GWAS

Case-control

65
Q

What is an imprinted gene

A

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

66
Q

Explain how chromosome rearrangement can lead to imprinting disorders

A

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

One will be lost

67
Q

Explain how aberrant methylation patterns (epimutation) can lead to imprinting disorders

A

Imbalance in methylation patterns can mean overactivation or underactivation

68
Q

Explain how uniparental disomy can lead to imprinting disorders

A

There may be double paternal or double maternal patterns = imbalance

69
Q

What is the conflict hypothesis

A

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

70
Q

Describe Prader-Willi syndrome

A

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
71
Q

Describe Angelman syndrome

A

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

72
Q

What and how can we detect PW deletions

A

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

73
Q

What is the transcriptome

A

Complete set of transcripts (RNA) expressed in a sample/tissue at specific point of time

Tissue specificity

Changes in response to stimuli/disease

74
Q

What post-transcriptional modifications enhance complexity in our genes

A

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

75
Q

What does alternative splicing achieve

A

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

76
Q

Describe splicing mechanisms

A

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

77
Q

Describe aberrant splicing

A

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

78
Q

Describe splicing mutations

A

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

79
Q

Describe the splicing mutation disease within the LMNA gene

A

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)
80
Q

Describe the methods for functional testing of predicted splice variants

A

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

81
Q

Describe the use of RT-PCR in beta thalassemia and leigh syndrome

A

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

82
Q

Describe when the minigene assay is use to analyse RNA

A

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

83
Q

Discuss why WES and WGS is not ideal in analysing RNA

A

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

84
Q

Describe transcriptome analysis

A

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

85
Q

Describe RNA sequencing library prep

A

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

86
Q

Describe the use of RNAseq in routine diagnostics

A

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

Human Medical Genetics (9 decks)

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