Epigenetics and Disease inc. X-inactivation Flashcards
1
Q
Review the current range of genetic diagnostic services for diseases where epigenetic mechanisms play a role in disease pathogenesis.
A
- Introduction and line of argument
- e.g. epigenetics is vital to noraml gene function
- epigenetic abnormalities are a major contributor to Human disease
- What is epigenetics and it’s normal function
- What are the mechanisms of epigenetics
- What disease are caused by epigenetics
- X-linked diseases (methylation/lyonisation)
- Imprinted diseases (eg PWS, AS, BWS, RSS, Temple and Wang syndromes),
- Diseases mediated by positional effects (eg FSHD),
- Diseases caused by genes that effect DNA modification (eg MECP2),
- Chromatin modelling disorders (e.g. Kabuki, Sotos, etc )
- Diseases mediated by non-coding RNAs (eg snRNAs; PWS/AS, BWS).
- Methylation in cancer
- What diagnostic services are avaiable for these diseases
2
Q
Definition of epigenetics
A
- Epigenetics refers to
- heritable and transient
- changes in gene expression
- that do not alter the primary DNA sequence
3
Q
Normal function of epigenetics
A
- Essential for development and many normal cellular processes
- Helps maintain genome stability and prevent illegitimate recombination
- Role in determining the conformation of chromatin
- Regulates the switching of genes on or off which determines how proteins are transcribed
- Contributes to variable expression of genes in different cell types
- X-inactivation and imprinting, both result in monoallelic gene expression are vital for normal development
- cell to daughter cell heritability, but not from parent to child
4
Q
List the mechanisms of epigenetics
A
- Epigenetic modifications to DNA are initiated and sustained by at least three mechanisms
- DNA methylation
- histone modification
- RNA-associated silencing
5
Q
Describe how DNA-methylation exherts an effect
A
- 5MeC pairs with guanine in the same way as unmodified cytosine but the methyl group acts as a signal recognised by specific MeCpG-binding proteins.
- These can then recruit other proteins associated with repressive structures such as histone deacetylases and have a role in regulating chromatin structure and gene expression.
- In contrast, demethylation relaxes chromatin allowing histone acetylation and binding of transcriptional complexes.
- Humans have 5 MeCpG-binding proteins:
- MBD1-4
- MECP2
6
Q
Describe how DNA is methylated
A
- Almost entirely restricted to cytosines that lie immediately 5’ of guanines in CpG dinucleotides - results in two methylated cytosines diagonal to each other on opposing DNA strands.
- In mammals ~70% of all CpG dinucleotides are methylated.
- Carried out by DNA methyltransferase (DNMT) enzymes - uses S-adenosylmethionine (SAM) as the methyl donor (results in S-adenosylhomocytosine)
- Animals deficient in DNA methyltransferase activity die at various stages of development.
- Added methyl group acts as a signal that is recognised by specific MeCpG-binding proteins.
7
Q
Where are CpG dinucleotides enriched in the genome?
A
- Concentrated on repetitive sequences
- characteristic of pericentric heterochromatin and dispersed transposons
- Also sporadically distributed in genes and intergenic sequences
- A high proportion gene promoter CpGs (known as CpG islands) stay unmethylated and so are less prone to deamination.
8
Q
Describe how DNA-methylation leads to increased mutation rate
A
- Cytosine targeted for methylation at C5 is vulnerable to deamination, producing
- 5MeC = thymidine
- cytosine = uracil
- Are differentially recognized by DNA repair enzymes
- hence high CpG mutation rate
- Deamination of 5MeC results in mutation of CpG to TpG and CpA on the sense and antisense strands, respectively.
9
Q
Describe how methylation patterns are maintained from cell to daughter cell
A
- Methylation patterns maintained by a specific methylase (DNMT1) to recognize a hemimethylated target (methylated on one strand)
- Once both strands are methylated they separate and act as templates during DNA duplication producing unmethylated daughter strands.
- These daughter duplexes will now provide new hemimethylated targets for continuing the same pattern of methylation.
10
Q
Describe the enxymes involved in methylation
A
- DNMT1
- DNMT3A
- DNMT3B
11
Q
Describe how DNA-methylation is vital for normal embryo development
A
- Though heritable, patterns of methylation are not fixed and rapid changes occur:
- Gametogenesis - substantial de novo methylated genomes in the sperm and egg.
- Early embryogenesis - wave of genome-wide demethylation at the pre-implantation stage. This epigenetic reprogramming erases adult methylation patterns.
- Post-implantation - large-scale de novo methylation by DNMT3A and DNMT3B.
12
Q
Describe how the position of methylation influences relationship to gene control
A
- In immediate vicinity of transcriptional start site
- blocks initiation
- In gene body
- does not block and may stimulate transcription elongation. May effect splicing.
- In repeat regions
- important for chromosomal stability
13
Q
Describe how histone modification exherts an epigenetic effect
A
- Histones are proteins that are the primary components of chromatin- the complex of DNA and proteins that make up chromosomes (see notes on DNA structure).
- The N-terminus of histone molecules protrude from the body of nucleosomes.
- Chemical modifications of amino acids in these ‘histone tails’ are major determinants of chromatin conformation
- This consequently influences DNA transcription
- In a non-compact form chromatin is active and the associated DNA can be transcribed.
- If chromatin is condensed (inactive), DNA transcription does not occur.
14
Q
Describe the different ways histones are epigenetically modified
A
- There are two main ways histones can be modified:
- Acetylation/Deacetylation
- adds/removes an acetyl group (COCH3)
- to free amino groups of lysines or arginines of H3 tail
- Methylation/Demethylation
- adds/removes an methyl group (CH3)
- to free amino groups of lysines or arginines of H3 tail
- Acetylation/Deacetylation
15
Q
Describe how histones are Acetylated/Deacetylated
A
- Acetylation/deacetyleation is catalyzed by
- histone acetyltransferases (HATs)
- histone deacetylases (HDACs)
- Lysine acetylation almost always correlates with increased chromatin accessibility and transcriptional activity
- Deacetylation is generally associated with heterochromatin and represses transcription
16
Q
Describe how histones are methylated/demethylated
A
- Methylation is catalyzed by
- histone methyltransferases (HMTs) histone demethylases (HDMs)
- The effect of this depends upon which residue is methylated and protein in which the modified histone is found
- For example
- methylation of a particular lysine (K9) on a specific histone (H3)
- marks silent DNA and is widely distributed throughout heterochromatin
- this is the type of epigenetic change that is responsible for the inactivation of the second X chromosome in females
17
Q
Describe how non-coding RNAs have an epigenetic effect
A
- Non-coding RNAs which have an epigenetic effect comes in at least 2 classes
- long non-codings RNAs
- effect structural transformations of chromatin
- short non-coding RNAs
- target individual mRNA’s for degredation
- long non-codings RNAs
18
Q
Describe how short non-coding RNAs have an epigenetic effect
A
- MicroRNAs, small strands of RNA ~22 nucleotides long, interfere with gene expression at the level of translation
- i.e. they regulate the translation of RNA transcripts into amino acid chains
- MicroRNAs form active ribonuclear complexes with cytoplasmic proteins.
- These complexes have RNAase activity.
- Each microRNA has a base sequence that is complementary to a specific messenger RNA (mRNA) sequence, meaning that each microRNA degrades a specific mRNA.
- 1 microRNA can degrade multiple mRNAs corresponding to several different genes, often with similar functions.
- Thus, microRNAs differ from RNAase enzymes in that the former are a targeted regulatory mechanism to reduce gene expression.
- MicroRNAs work post-transcriptionally by binding to the 3′-untranslated regions of their target mRNAs, thereby inducing enzymatic degradation and preventing translation.
19
Q
Describe how long non-coding RNAs have an epigenetic effect
A
- These transcripts are ~200 bp long and are thought to form ribonucleoprotein complexes
- that interact with chromatin, regulating histone modifications and the structural transformations that distinguish heterochromatin from euchromatin.
- Previously, lncRNA was thought to be a by-product of normal gene transcription
- but we now know that lncRNAs show cell type-specific expression and also respond to diverse environmental stimuli
- suggesting that their expression is both regulated by and responsive to the environment
20
Q
Describe the types of diseases which are mediated by epigenetic mechanisms
A
- X-linked diseases
- Imprinted diseases
- Diseases mediated by positional effects
- Diseases mediated by STR expansion
- Diseases caused by genes that effect DNA modification
- Chromatin modelling disorders
- Diseases mediated by non-coding RNAs
- Cancer
21
Q
Describe why some x-linked disorders are mediated by epigenetic mechanisms.
A
- A difference in sex chromosome complement between male and female requires a mechanism to bring about equal gene expression of X linked genes
- X inactivation is an epigenetic process causing differential gene expression and chromatin changes between the two X chromosomes of a normal female.
- It results in transcriptional silencing of one X in a female, resulting in both sexes having only one functional copy of X
- XLR diseases may be expressed in carrier females where there is X-inactivation skewing towards the wilt-type X
- ABCRs are typically assymptomatic but may lead to adnormal phenotypes in females with a t(X:A)
22
Q
Describe some examples of X-linked diseases where x-inactivation skewing leads to disease.
A
- DMD
- random skewing leads to unmasking of point mutation causes manifesting female
- t(X:A) in DMD gene leads to skewing and silencing of der(X) and unmasking of point mutation on normal X
23
Q
Describe the diagnostic tests for investigating X-inactivation.
A
- Replication banding
- A cytogenetic technique to determine which X is inactivated, for example in an X;Autosome translocation. Analysis of sufficient metaphases can allow calculation of a ratio of any skewed inactivation
- Methylation specific PCR
- bisulphite modification of DNA
- methyl and non-methly specific primers give different fragment sizes
- relative intensity measured on ABI
- HUMARA Assay
- methy-specific digestion of DNA
- PCR across AR gene STR
- relative intensity of two allele sizes measured on ABI
24
Q
Describe how imprinting disorders are mediated through epigenetic effects
A
- Genomic imprinting is an epigenetic phenomenon that causes genes to be expressed in a parent-of-origin-specific manner
- In the developing sperm a paternal imprint is established, whereas in developing oocytes a maternal imprint is established
- Gene expression occurs from only one allele of imprinted genes
- The major mechanisms that are involved in establishing the imprint are DNA methylation and histone modifications
- Imprinting disorders occur when the functional dosage of an imprinted loci deviates from its natural level of one copy
- many mechanisms may cause this e.g.
- abnormalities in establishing the imprint in the gametes
- deletions/duplications/mutations/UPD
25
Q
Describe examples of diseases caused by imprinted genes and the diagnostic tests available
A
- Prader-Willi /Angelman syndrome
- Beckwith-Wiedemann/ Russell-Silver syndrome
- Methylation sensitive MLPA often first line test for imprinting disorders
- Can detected abnormal imprinting pattern at disease loci and determine if the mechanism is due to a deletion
- If normal then other mechanisms explored
- Involves a mix of karytype for ABCRs, sequencing for point mutations, microsatellite analysis for UPD etc
26
Q
Describe how epigenetics can lead to diseases mediated by position effects and the diagnostic tests available for this disorder
A
- A D4Z4 repeat at 4q35 has a normal number of repeat units is 11-100 (38kb- 300Kb)
- DUX4 gene located at 4qter distal to repeat is normally repressed by epigenetic silencing (hypermethylation)
- A contraction of the D4Z4 repeat to 1-10 units leads to hypomethylation
- The position effect of the DUX4 gene to proximal genetic elements leads to expression
- DUX4 expression causes apoptosis of myoblasts leading to FSHD phenotype
- Southern blot is hybridized with a D4Z4 probe in order to determine the size of the D4Z4 repeat and infer methylation status
27
Q
Describe how epigenetics leads to diseases mediated by STR expansion. What tests are available for this disorder?
A
- Fragile X syndrome is caused by expansion of a CGG trnucleotide repeat in the promotor of the FMR1 gene
- Expansions >200 become hyper-mthylated at the repeat site and also at a CpG upstream of FMR1, leading to transcriptional silencing
- Front line testing involves simple f-pcr across expansion but this will not detect methylation status of repeat
- Southern blotting with methylation specific enzymes can detect methylation status of expansion and potential X-skewing in females, but laborious
- methy-specific PCR assays quicker alternative to detect methylation status of repeat but won’t detect skewing.
28
Q
Describe diseases caused by genes that effect DNA modification
A
- Humans have 5 MeCpG-binding proteins: MBD1-4 and MECP2
- Loss-of-function of MECP2 causes Rett syndrome
- MECP2 function is needed in mature neurons.
- Rett is an X-linked condition, the absence of MECP2 from neurones that have inactivated the normal X chromosome means some signals are not read correctly
- leads to neurodevelopmental disorder of females and lethality in males
- Arrested development 6-18 months. Small hands/feet and deceleration of rate of head growth
29
Q
Describe the genetic tests available for RETT syndrome
A
- Diagnostic testing involves sequencing plus copy number analysis of the MECP2 gene
30
Q
Describe diseases caused by chromatin modelling disorders and the genetic tests available for diagnosis
A
- Sotos syndrome
- Cerebral gigantism, MR, behavioural problems
- LOF mutations in NSD1, which is a histone methyltransferase that can negatively and positively influence transcription
- Kabuki syndrome
- Distinctive face, mild to severe developmental delay and ID
- LOF mutations in
- KMT2D = methyltransferase
- KDM6A genes = de-methylase
- Sequencign and MLPA for mutations
31
Q
Describe epigenetic diseases mediated by non-coding RNAs and diagnostic test available
A
32
Q
Describe epigenetic causes of cancer
A
- Lynch Syndrome
- somatic methylation of the MLH1 promoter leading to silencing of the gene
- deletions in the 3’ end of EPCAM upstream of MSH2 resulting in epigenetic hypermethylation of the MSH2 promoter and loss of MSH2 expression
- MS-MLPA of MLH1 promoter
- MLPA for EPCAM deletion
33
Q
Describe epigenetic causes of response to treatment in cancer
A
- Alkylating agents are limited when in the presence of the DNA-repair enzyme MGMT (methyltransferase)
- Cross-linking of double-stranded DNA by alkylating agents is inhibited by the cellular DNA-repair mechanism, MGMT.
- MGMT promoter methylation causes cells no longer produce MGMT
- Cell are then more responsive to alkylating agent therapy
- Methylation of the MGMT promoter in gliomas is a useful predictor of the responsiveness of tumors to alkylating agents
