Path - Genetics - Exam 3 Flashcards
(85 cards)
1
Q
How can single gene recessive/dominant disorders occur?
A
- Recessive:
- homozygous
- compound heterozygous - Dominant:
- heterozygous inherested
- de novo
2
Q
Explain the standard patterns of mendelian inheritance
A
The modes of Mendelian inheritance are autosomal dominant, autosomal recessive, X-linked dominant, and X-linked recessive.
- Autosomal dominant: 50 percent chance of having an affected child with one mutated gene (dominant gene) and a 50 percent chance of having an unaffected child with two normal genes (recessive genes).
- i.e. huntingtons – CAG repeat in HD gene on chromosome 4 - Autosomal Recessive – need two carrier parents
- X-Linked Dominant: X-linked dominant traits do not necessarily affect males more than females (unlike X-linked recessive traits). The exact pattern of inheritance varies, depending on whether the father or the mother has the trait of interest. All daughters of an affected father will also be affected but none of his sons will be affected (unless the mother is also affected). In addition, the mother of an affected son is also affected (but not necessarily the other way round).
- X-Linked Recessive - X-linked recessive inheritance is a mode of inheritance in which a mutation in a gene on the X chromosome causes the phenotype to be expressed in males (who are necessarily hemizygous for the gene mutation because they have one X and one Y chromosome) and in females who are homozygous for the gene mutation, see zygosity.
3
Q
Explain the cell biological basis of mendelian inheritance
A
- loss of function – much more common and more likely to be recessive
- gain of function - more likely to be dominant
- biallelic – both in phenotype.
- dosage sensitive or insensitive
- monoallelic – one will be silent
- dosage sensitive
- maternal or paternal
a) M/P Allelic Expression – blocks other allele
b) M/P Imprinting – blocking itself
For a loss of function mutation:
- heterozygotes for dosage insensitive gene will be phenotypically normal = RECESSIVE
- heterozygotes for dosage sensitive genes are haploinsufficient. = DOMINANT.
- can be increase or decrease in dosage
- i.e. Charcot-Marie-Tooth Disease – a duplication leads to 3 alleles. Nerve condition.
Haploinsufficiency is the phenomenon where a diploid organism has only a single functional copy of a gene – not enough protein is made.
4
Q
What is the molecular basis of dominance?
A
- Gene is “dosage” sensitive
A. reduced gene dosage, expression, or protein activity (haploinsufficiency)
B. increased gene dosage - Gene mutation causes “gain of function”
A. Ectopic or temporally altered mRNA expression
B. Increased protein activity
C. Toxic protein alterations
D. Altered structural proteins
E. Dominant negative effects
F. New protein function
5
Q
Define Penetrance and Expressivity:
A
Penetrance: – Proportion of individuals with a mutation that develop disease = no. of people with disease/no. of people with mutation.
I.e. BRCA1. Appears recessive, but is actually just low penetrance.
Expressivity:
Given that the disease is present, this refers to the degree to which the disease is expressed (number and severity of clinical features). Genetic and environmental factors.
6
Q
Explain the biological basis for variable penetration and expressivity (modifiers of mendelian inheritance)
A
Variable penetration – gene is penetrative in some and non-penetrative in others. Expressivity – severity.
Why the difference?
- Stochastic (random probability of distribution, i.e. required ‘second hit’)
- environmental
- genetic – variants in other genes
- heterogenetic?
- Constitutional or mosaic?
I.e. Autosomal Dominant polycystic kidney disease (APKD).
7
Q
What is Genotype-phenotype correlation?
A
- like between specific genetic mutation (genotype) and disease characteristics (phenotype)
- observed genotype-phenotype correlations can sometimes be explained by considering the specific effect that the genotype has on consequent or residual protein function
- genotype-phenotype correlation is not always observed – other factors affecting gene expression may interfere, i.e.:
- modifier genes
- chance effects
- environment
8
Q
/What is Duchenne muscular dystrophy (DMD) and what causes it?
A
- x-linked, therefore hemizygous males and homozygous females
- wheelchair dependent by 12
- cause: mutations in DMD gene which encode dystrophin – structural protein – muscle cell membrane.
- Deletions that result in frame-shifts – produce unstable mRNA and no dystrophin protein
- If deletion occurs but no Frameshift – becker muscular dystrophy (milder)
9
Q
/What is AATD and what causes it?
A
- alpha1-antitrypsin (protease inhibitor) deficiency
- increased risk of liver and lung disease
- due to:
a) missense mutations – results in ATT polymer formation (bad)
b) null alleles (nonsense or Frameshift) - AAT deficiency but no polymer formation so no risk of liver disease.
10
Q
What is Mosaicism?
A
Post-zygotic mutations resulting in two or more genetically distinct cell lines.
If it arises later – more localized.
Phenotype will depend on the proportion of mutant cells.
Typically results in milder disease
EXAMPLE: Monosomy X (turner syndrome)
11
Q
What is Mono-allelic and bi-allelic expression ?
A
biallelic – both in phenotype. A + a
- dosage sensitive or insensitive
monoallelic – one will be silent: A OR a
- dosage sensitive
- dependent on parent of origin OR random
- Dependent on parent of origin:
a) M/P Allelic Expression – blocks other allele??
b) *M/P Imprinting – blocking itself - Random:
c) X-inactivation
d) autosomal monoallelic gene expression
12
Q
- How does dosage affect clinical outcomes? What is haploinsufficiency? Vs Dominant Negative?
A
For a loss of function mutation:
- heterozygotes for dosage insensitive gene will be phenotypically normal = RECESSIVE
- heterozygotes for dosage sensitive genes are haploinsufficient. = DOMINANT.
Haploinsufficiency is the phenomenon where a diploid organism has only a single functional copy of a gene and it is not tolerated:
- insufficient protein hypothesis
- balance hypothesis (imbalance of protein complex subunits)
Vs Dominant Negative – protein loss of normal function that interfered with normal function of other proteins
Gene functions that are inherently dosage sensitive:
- products that are rate-limiting
- competitive products that determine a developmental or metabolic switch
- fixed stoichiometry
EXAMPLE: autosomal dominant osteogenesis imperfect – heterozygous loss of function
a) mild – haploinsufficiency – no alpha chain
b) severe – dominant negative – abnormal alpha chain production
13
Q
What is Imprinting?
A
Gene expression according to parent of origin:
- maternal: allele from mother switched off
- paternal: allele from father switched off
Reset each generation
EXAMPLE: Prader-Willi syndrome
14
Q
What is X-inactivation?
A
Steps:
- X chromosomes counted
- Xist is transcribed from the future inactivated X chromosome/s
- Xist RNA then coats the body of the chromosome
- Expression of Xist is considered necessary and sufficient for inactivation; it recruits other silencing proteins
- usually random
- XCl ensures correct dosage of X-linked genes – some genes on X chromosome escape inactivation (pseudoautosomal regions, need 2n)
- Human females = ‘functionally mosaic’ as 2 different cell lines differentiated by which X chromosome is expressing
- EXAMPLE: DMD in females – some muscle fibers stain positively for dystrophin, others not.
15
Q
What is skewed vs random X-inactivation?
A
Usually random, but may be skewed towards non-mutant chromosome, therefore could explain phenotypic variability in females.
EXAMPLE: DMD – mutant allele favored, therefore most cells mutant.
16
Q
Explain characteristics of genetic variation in human populations (small and large scale genomic variants)
A
- low mutation rate, we each have 8-12 new variants
- most variants therefore inherited
- ~1:8 of all base positions in protein coding exome have been observed to vary
- many variants not functionally important and occur at minor allele frequencies (MAF) of >0.1% or 1% in the population. If >1% - benign variation (polymorphism). As opposed to pathogenic variation
Small scale (bp):
- substitutions
- base deletions
- base insertions
Large scale (kb-Mb)
- insertions
- deletions
- inversions
Genome scale:
- chromosomal aneuploidy (wrong number of chromosomes)
- polyploidy (wrong number of sets)
17
Q
Explain factors determining genetic population structure
A
Hardy-Weinberg principle:
If population is:
- large (therefore genetic ‘random’ drift minimized)
- randomly mating
- no migration
- no selection pressures on a particular genotype
- mutation rate remains constant – new mutation rate = rate of mutation loss
Then, genotype frequency will remain unchanged from one generation to the next.
NAME:
- mutation rates
- selection
- migration
- random drift
18
Q
Explain frequency of genetic diseases in populations
A
p + q = 1 (q = recessive allele frequency)
P2 + 2pq + q2 = 1
19
Q
Explain history of uses and misuses of genetics in medicine
A
- genetic determinism
- genetic discrimination
- Future: genetic cloning – what is perfect?
20
Q
Explain deviations from random mating - assortive mating; consanguinity
A
Assortive mating: individuals with similar genotypes and/or phenotypes mate with one another more frequently than would be expected under a random mating pattern.
Consanguineous union: second cousins or closer
Coefficient of inbreeding (F) = P (two alleles identical by decent)
For first cousins, spouses share 1/8 of genes, therefore progeny autozygous at 1/16 of loci, therefore F=0.0625.
Coefficient of Relationship (R) = proportion of genes that consanguineous spouses share as a result of common decent.
R = 2F, therefore R = 1/8 for first cousins.
Siblings: R = 1/2 and F = 1/4.
First: R= 1/8 and F =1/16
Second: R = 1/32 and F =1/64
21
Q
What causes variation?
A
- DNA repair mechanism to contend with damage due to environmental agents
- DNA replication errors
- Homologous DNA recombination during meiosis
- Retrotransposition (regions of DNA that can copy and reinsert themselves somewhere else)
Compared to ‘reference genome’
22
Q
What are the consequences of variation?
A
- no change in phenotype
- alternative phenotypes of no medical consequence
- disease susceptibility
- pathogenic
23
Q
What is the difference between DNA damage and mutation?
A
Damage = physical change in structure of DNA
- endogenous or exogenous
Mutation = change in the base sequence of DNA
24
Q
What factors can cause DNA damage?
A
a) exogenous factors, i.e.
- UV light
- ionizing radiation
- chemical agents - human mad (alkylating agents, heavy metals tobacco etc), chemo, medical exposures
b) endogenous factors, i.e.
- cellular metabolism
- replication errors
25
How does ionizing radiation induce DNA damage?
- Unique feature: clustered damage that can result in DSBs
- Degree/type of damage related to energy level:
a) low energy – isolated lesions
b) high energy – clustered damage
- bystander damage to surrounding cells
- E.g. – Chernobyl. Increased frequency of chromosomal rearrangements in thyroid cancer – consistent with DSBs + subsequent repair resulting in fusion genes.
26
How do alkylating agents induce DNA damage?
Sources:
- certain industries
- smokers
- HCPs with chemo
Cause direct damage to DNA by adding an alkyl group to N and O atoms of purines and pyrimidines.
Consequences:
- base mismatches (i.e. methyl group to guanine)
- crosslinking
- strand breaks
27
What are the possible cell responses to DNA damage?
a) Cell cycle arrest – until repair
b) DNA repair – independent of cycle
c) Apoptosis – irretrievable damage
d) Transcription – induction of proteins and ncRNA involved in cell cycle, signaling and DNA repair)
28
What are the types of DNA damage?
- base loss
- base modification
- inter-strand X-links
- DNA protein C-links
- Strand breaks
29
What are the types and mechanisms of DNA repair?
1. Single strand breaks
a) base excision repair
- glycosylases sense damage and break the sugar-base done.
- short patch or long patch
b) nucleotide extension repair
- corrects thymine dimers and other large chemical adducts
- i) global (anywhere in genome)
- ii) transcription coupled (at actively transcribed regions)
- removes large patch around error, so good for repair of bulky lesions
c) mismatch repair
- repairs mismatched and insertion/deletion loops that are created during DNA replications
- errors sensed by mismatch proteins
- nicks strand at closes GATC site to error
2. Double strand breaks:
No template to use
a) non-homologous end joining
- G0 and G1 phase
- protein complexes assemble at broken ends and rejoin them using ligases, regardless of sequence
b) homologous recombination
- S phase (synthesis dependent – sister chromatid)
- single strand from damaged DNA invade the undamaged homologue, which is used as a template for repair
30
Where are DNA damage checkpoints in the cell cycle and how do they work?
- designed to detect DNA damage, replication errors and spindle defects and arrest cell cycle to facilitate repair
- checkpoint activation controlled by two master kinases: ATM and ATR
a) G2/M: unreplicated or damaged DNA?
b) G1/S: damaged DNA?
c) Intra-S: Damaged DNA, replication errors or incomplete replication?
31
Describe examples of diseases caused by defects of DNA repair and replication
Xeroderma Pigmentosum:
- acute susceptibility to UV light-induced skin cancer due to mutations in genes involved in nucleotide excision repair
Li-Fraumeni Syndrome:
- AD cancer predisposition syndrome
- Cancer predisposition syndrome
- Loss of function mutations in TP53 gene
Lynch Syndrome:
- AD cancer predisposition syndrome
- Increased risk of colon cancer + more
- Loff of function mutations in MLH1, MSH2, MSH6, PMS2
32
Appreciate how cancer therapeutics manipulate DNA damage and repair mechanisms
Mechanism:
a) direct DNA damage (i.e. radiation, alkylating agent)
b) inhibition of DNA replication/repair
Resistance can occur through increased DNA repair.
Negative consequences:
- drug toxicity, i.e. untargeted effects
- teratogenicity – affects growth of embryo/feotus
33
Briefly define different types of genetic testing
1. Diagnostic – confirms diagnosis in an affected person, and may be prognostic as well
2. Predictive – testing of unaffected individuals, usually in the absence of corroborating evidence, for:
- pre-symptomatic (will they develop the disease?)
- carrier
- prenatal/pre implantation
3. Screening – testing of individuals at population-level risk of disease (i.e. newborn), where there is an advantage of detecting conditions early
4. Somatic – testing of tumor tissue (not germline) for:
- diagnostic
- monitoring
- choice of therapeutic agents
5. Pharmacogenetic – metabolism/drug toxicity
34
Be aware of appropriate sample sources for genetic testing
1. Peripheral blood (whole blood)
- DNA extracted from WBCs
- preferred source of DNA for testing – plentiful and easy to access, minimally invasive
2. Buccal cells, urine, skin
- collected by swab inside cheek, early morning urine or biopsy
- used as a second source of DNA is suspicion of mosaicism,
- used for comparison of tumor DNA to constitutional DNA, OR
- used when it is difficult to get blood for some reason
3. Chorionic villus Sampling (placenta, 11-14 weeks) and Amniocentesis (amniotic fluid, 15-18 weeks)
- invasive, therefore risks of bleeding, infection, loss of pregnancy and incorrect results due to contamination with maternal cells
- used for chromosomal or molecular testing
- used for studying metabolic diseases
4. Cell free DNA (cfDNA)
- DNA not contained by membranes but floating free in circulation can be isolated
- Sources of cfDNA:
- haematopoeitic cells
- foetal cells
- tumor cells
- used for non-invasive prenatal screening – aneuploidies, can be used early in pregnancy
- used for tumor detection and monitoring – not widely available
35
How do we choose what to test within the DNA sample?
Depends on:
1. Change to be detected
- size
- where it occurs
- type of change
2. Volume of samples being tested
3. Purpose of testing (known or unknown mutation). If you know where it is, focus on that region on which the variant occurs, i.e. Sanger sequencing of a single exon.
36
Explain the difference between targeted and untargeted genetic testing
Targeted – specific alleles of copy number variants are tested for
Untargeted – whole or multiple genes, whole genome. More than one variant may be found – which is the mutation?
37
Appreciate the key factors in determining whether a genetic variant is disease-causing or benign
5 tier classification system:
```
Class 1 - Benign
Class 2 – Likely Benign
Class 3 – Uncertain significance
Class 4 – Likely pathogenic
Class 5 – Pathogenic
```
Look to:
- public resources – literature and databse
- in silico prediction – algorithms
- Key, powerful factors:
- segregation of genotype with disease
- functional studies
Segregation of genotype:
- testing of proband’s parents usually recommended to test this
- based on family history and inheritance pattern expected to be observed
- AD: i. de novo – neither parent
ii. Inherited – an affected parent should have variant
- AR: both parents should be carriers
RNA and functional studies:
- focus on dynamic effects of changes at the DNA level on gene transcription, translation and protein-protein interaction
- tests on cells, animal models etc
38
Understand the concept of clinical utility, using example clinical scenarios
How can genetic testing actually improve patient management?
Ask yourself:
- why am I requesting this test?
- how does it help my patient? BRCA genes – can get prophylactic surgery, but there is nothing you can do about Huntington’s.
- can the result harm my patient or their family?
- is there a better approach or test I could use?
- could I be using resources more wisely?
- Eg – full genome testing as last resort
39
What are some of the ethical challenges of testing?
- may affect life choices
- may affect continuation of pregnancy
- access to insurance
- implications to extended family
EG – one family member of 2 cousins is congenitally deaf (unknown cause, often recessive mutation). Another 2 are going to get married and what children – should they be tested? Is deafness a real problem? Will knowing the risks make a difference? Will knowing the mutation make a difference to the deaf child? Should the deaf child be tested for the benefit of her aunt/uncles future children? Etc
40
What is the key info about Huntington’s Disease in relation to genetic testing, including symptoms, mechanism, tests involved and clinical utility?
Symptoms:
- mood swings/irritability
- depression
- anger
- forgetfulness
- restlessness etc
About:
- autosomal dominant
- caused by triplet repeat expansion – dynamic mutations (increased no. of repeats within a repetitive sequence)
- CAG repeat 40 will result in HD
- complete penetrance
- ½ chance of inheriting it from parents if one has disease
How are CAG repeats tested?
- PCR primers flank repeat region
- Amplify fragment
- Determine length and subtract non-repetitive sequence to determine number of CAG repeats
- Test proband first
- Two independent samples should be collected
Should people be tested?
- Untreatable and fatal
- BUT good medical care makes a big difference
- If they have children, children might want to know their risks, i.e. predictive testing.
- BUT predictive testing can have extreme life-changing consequences, i.e. insurance, psych etc.
- IS CRUCIAL to confirm family disease really is HD
- Why? Imagine child’s result is negative – they are reassured. But years later they develop similar symptoms – might have been a different disease
- Predictive testing must be supported by appropriate genetic counseling
41
What is the key info about MCAP/PROS in relation to genetic testing, including symptoms, mechanism, tests involved and clinical utility?
Symptoms:
- megalencephaly
- digital anomalies
- CT dysplasia etc
Causes by:
- post-zygotic mutation - mosaicism
Tests:
- Sanger sequencing can tell you the two alleles you inherited from mum and dad, but not the proportion of each in your cells
- Massively parallel sequencing can
- If germline heterozygous, there should be roughly 50/50. It may be important for parents to known if it was heritable so they know their risks for future children – negligible if post-zygotic mosaicism.
- Important for parents
- Need to test clinically affected tissue samples – dermal biopsy preferred overlying an affected area
42
What are the inherent risks of prenatal testing and how can some of them be overcome?
- often to available corroborating evidence that the foetus is affected
- contamination with maternal cells – risk that sample will actually reflect maternal genotype
- sample mix-up
qfPCR STR analysis:
- uses tandem repetitive sequence to identify maternal and paternal alleles
- should see allele from both mum and dad
43
What would you expect to see if foetal sample is contaminated with maternal DNA?
Note: mother and foetus should share one allele at each location – this confirms foetal sample identity
If contaminated we see:
a) a third peak will appear in the foetal analysis that is identical to the mother’s second allele
b) skewing of the peaks (maternal one bigger), indicating that there is more of maternal allele present than paternal
44
What would you suspect if a child had developmental delay + additional congenital abnormalities or dysmorphic features? What test would you use, what will it tell you and how does it work?
Chromosomal anomaly (i.e. multiple gene deletion syndrome = Williams-Beuren syndrome)
Take blood sample. Test = SNP microarray analysis. This will give info on deletions and duplications across the whole genome at high resolution.
Tests ~300,000 markers using fluorescence to determine:
- copy number
- zygosity
45
/What is the clinical utility of knowing a child has Williams-Beuren syndrome?
Can test for/monitor related consequences:
- cardiology evaluation
- calcium determination
- thyroid function
- glucose tolerance
Assessment/support for developmental/behavioral issues
46
What is the difference between molecular genetics and cytogenetics?
Molecular genetics:
- primarily concerned with interrelationship between DNA, RNA and synthesis of polypeptides
- molecular genetic tests typically DNA or RNA based
Cytogenetics:
- primarily concerned with structure, properties and behavior of chromosomes
- tests typically chromosome based
47
What is the difference between genome and epigenome?
Genome = complete genetic info.
Epigenome = multitude of chemical compounds that can tell the genome what to do. I.e. histones, DNA and chemical tags around it - 2nd layer of structure. It shapes the physical structure of the genome. It tightly wraps inactive genes, making them unreadable.
Genome is for life, but Epigenome is flexible. Tags react to outside world, i.e. diet and stress, and adjusts genes.
48
What is epigenetics?
- primarily concerned with heritable changes in gene expression that don’t involve changed to underlying DNA sequence – a change in phenotype without a change in genotype.
- Tests limited to detection of DNA methylation, but epigenetic mechanisms also include histone modifications and non-coding RNA
- Can be influenced by age, environment, lifestyle and disease state
49
Define congenital, genetic and inherited diseases.
Congenital disease – present at birth. Doesn’t have to be genetic as it could happen in utero.
Genetic disease – caused by chromosome or gene defects (cytogenetic vs molecular genetics). Also doesn’t have to be inherited because new mutations can occur. I.e. de novo.
Inherited disease – passed from parent to offspring. Doesn’t need to be congenital - it can be an adult onset disease.
- caused by genetic abnormality transmitted from parent to offspring. Can have de novo (new) genetic abnormality.
50
What are the broad categories of genetic disorders?
a) Monogenic
- change/s in one gene sufficient for disease.
- single gene can cause single disorder or multiple
- a mutation in one of many genes can cause a single disorder. I.e. deafness can be caused by a change in one of many different genes.
b) Polygenic
- multiple genes contribute to phenotype
c) Complex/multifactorial
- can be caused by interaction between variants in multiple genes and the environment.
- don’t typically follow recognizable inheritance patterns but do seem to run in the family.
- can’t really test for it
- example: type 2 diabetes 4% heritable.
51
What is the difference between a variant and mutation?
Variant = any change
Mutation – either any change or specifically a change that causes a disease. I.e. pathogenic variant.
52
What is a polymorphism?
variant that is common in normal population so presumed benign.
53
Why do genetic testing?
a) diagnostic - confirms diagnosis in an affected person and may provide prognostic info
b) predictive - testing of unaffected individuals
c) screening - testing of individuals at population-level risk of disease
d) somatic - testing of tumor tissue
e) pharmacogenetic - metabolism/drug toxicity
- predicts how an individual will respond to a particular medicine
54
Discuss diagnostic testing
- confirms diagnosis in an affected person and may provide prognostic info
- example – trisomy 21
- can be extension of clinical diagnosis. I.e. Long QT syndrome or Charcot Marie Tooth disease can test genotype problem which may guide understanding of prognosis and also best treatment options.
55
Discuss predictive testing
- testing of unaffected individuals (usually in the absence of evidence)
i) pre-symptomatic – determines whether a person will develop a disease
ii) Carrier (determines whether they can potentially have affected their offspring)
iii) prenatal/pre-implantation
Examples:
- neurodegenerative diseases (huntingtons, spinocerebellar ataxias, mendelian dementias). Assists life choices.
- familial cancer - breast/ovarian, colorectal. Cancer surveillance and risk reducing surgery.
- cardiac diseases – long QT syndromes, familial hypercholesterolaemia. Can get chemoprophylaxis implants.
- respiratory diseases – alpha1-antitrypsin deficiency – lifestyle choices.
56
What is the historic and current definition of gene?
Historical definition of gene: discrete unit of heredity, then distinct locus on a chromosome, sequence pattern etc.
Current definition: A gene is a locus (or region) of DNA that encodes a functional RNA or protein product. DNA segment that contributes to phenotype/function.
In the absence of a demonstrated function a gene may be characterized by sequence, transcription of homology. Not all genes produce proteins.
OR entire nucleic sequence necessary for the synthesis of a functional polypeptide or RNA.
They overlap, run in opposite directions and promoters can be distance from actual gene.
57
If there is a change in DNA, what effect will the resulting protein have on cell function?
- no effect
- apoptosis
- disease-causation
Examples:
Fragile X – triplet repeat expansion in FMR1. Gene is switched off.
Progeria – single base substitution in LMNA. Cryptic splice site and altered protein.
Cystic fibrosis – coding DNA change on CFTR/ Absent protein/impaired protein function.
58
In epigenetics, which 3 main mechanisms act by modifying chromatin or DNA?
a) DNA methylation
b) histone modification
c) non-coding RNA (ncRNA)
59
How does DNA methylation affect epigenetics?
Can get methylated cysteine sites, and this methylation is faithfully maintained with DNA replication.
Associated with repressed gene expression. Methylation of promoters is associated with no or low gene expression.
60
How does histone modification affect epigenetics?
Histone tails can be modified by addition/removal of chemical groups. Acetylation.
i) transcriptional repression – DNA access limited by transcription factors. HYPOACETYLATION.
ii) transcriptional activation – open formation. ACETYLATION CAUSES THIS as it weakens histamine tails.
61
How does non-coding RNA affect epigenetics?
Regulates gene expression by:
- promote mRNA degradation and inhibit translation
- alter chromatin configuration
- Example: X Chromosome inactivation. Only one X chromosome in cells is active, the other is a Barr Body (inactive). X inactivation is random, and is mediated by expression of non-coding RNA
- Xist transcribed from X chromosome, which then coats that chromosome and silences it and recruits other proteins to help silence it. This can explain disease expression in X-linked disorders.
62
From the above, what increases gene expression and what decreases it?
Gene expression increased by:
- unmethylated DNA
- histone acetylation
Repressed by:
- DNA methylation
- Histone hypoacetylation
- Chromosome silencing
63
What tests are used for genomic variation detection?
a) light microscopy
b) FISH
c) chromosomal microarray
d) QF-PCR - huge resolution of single locus
e) MLPA - even higher
f) Circulating cell-free DNA and molecular counting
g) whole genome sequencing
64
Discuss light microscopy.
Resolution:
65
Discuss FISH.
Fluorescence in situ hybridisation.
Resolution: ~200kb
- about >25x magnification of light microscope
Advantages:
- culture not required for interphase FISH - rapid analysis
- numerical and structural anomalies detected
Disadvantages:
- only small region interrogated
- careful design of experiment required
66
Discuss chromosomal microarray.
Resolution: ~50kb
| - about
67
What are the p and q arms of a chromosome?
```
p = short
q = long
```
68
Define metacentric, Submetacentric and Acrocentric
Metacentric – centromere divides chromosome into almost equal parts
Submetacentric – uneven
Acrocentric – almost missing short arm so it is a satellite.
69
How is a numerical abnormality represented?
47,XX,+22
| So there are 47 chromosomes in a female, there is an extra chromosomes 22.
70
How is a numerical abnormality represented?
46,XY,t(11;22)(q23.3;q11.2)
There are the correct amount of chromosomes in a male, but there is translocation between 11 and 22 chromosomes. One homologue of chromosome 11 and one homologue of chromosome 22. Second brackets describe break points in each chromosome.
71
What are the major types of chromosomal variants?
1. numerical - most result in miscarriage
2. structural
- chromosomal breakage and reunion in a different configuration
- Balanced = no net gain or loss (just rearrangement)
- Unbalanced – gain or loss of chromosomal material
3. somatic
- results in cell lineages with different chromosomal constitution in the same individual
72
What are the types of numerical variants?
a) Euploid (equal number) numerical chromosomal aberrations.
- triploid (3 of everything) = 3x23=69
- tetraploid (4 of everything) = 4x23=92
b) Aneuploid – only specific chromosomes, not all
- trisomy 21 – DS
- trisomy 18 – Edwards syndrome
- trisomy 13 – patau syndrome
- Monosomy X – turner syndrome
- 47,XXY – klinefelter syndrome
73
What are the types of structural variants?
a) Reciprocal translocation
b) Isochromosome
c) Ring chromosome - literally a ball
d) copy-number variations
74
Discuss Reciprocal translocation
Meiotic pairing and segregation.
- carriers phenotypically normal but can be problem for children
i) *Alternate chromosomes
ii) Adjacent I (11 and 22 still)
iii) adjacent II (11 paired with 11 and 22 with 22).
iv) *Tertiary aneuploidy (one gamete has 3 and the other has 1, but both have parts of chromosomes 11 and 22)
v) interchange aneuploidy – same as above but gamete with 3 lands up with all chromosomes 22 material.
- only alternate and tertiary aneuploidy compatible with life.
i.e. Robertsonian translocation – risk for live born child with translocation down syndrome. Child can be normal (even though you are not), be a normal carrier or get down syndrome.
75
Discuss Isochromosome
An unbalanced structural abnormality in which the arms of the chromosome are mirror images of each other i.e. p and p or q and q.
Example: tetrasomy 12 p – there are 4 of the p arms
76
Discuss Copy-number variations
Copy-number variations (CNVs) are a form of structural variation that manifest as deletions or duplications in the genome.
For example, the chromosome that normally has sections in order as A-B-C-D might instead have sections A-B-C-C-D (a duplication of "C") or A-B-D (a deletion of "C").
- Results in structural chromosomal rearrangement and copy number variation.
- can’t see under microscope but can under microarray
- Example: DiGeorge/Velocardiofacial syndrome
77
Discuss somatic variations.
- results in cell lineages with different chromosomal constitution in the same individual
- Results in:
a) haematological malignancies
b) solid tumors
c) mosaics (mixture of cells of more than one genoytype)/chimeras (organism is composed of cells from different zygotes)
78
What are some examples of somatic variations?
a) Acute Myeloid Leukemia – if it is due to translocation, more favorable prognosis and specific treatment with ATRA
b) ALK gene re-arrangement
- gene re-arrangement found in young non-smoker with non-smalll cell lung carcinoma.
- patients with ALK gene re-arrangement have better overall survival when treated with crizotinib
79
Briefly explain sanger sequencing
Sanger sequencing is a method of DNA sequencing based on the selective incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication.
You have a dye for each A,G,C,T. Each die will stop at that particular nucleotide. Do this a whole bunch of times and arrange chains in order from shortest to longest so you can see the sequence of terminating nucleotides.
The translation machinery is equipped to choose the correct one out of three reading frames. As translation initiation codon is ATG.
80
What are the disease-causing molecular changes?
a) Substitution
i) Missense – one AA is changed
ii) Nonsense – AA changed to make a stop codon
iii) Frameshift – deletion/insertion that shifts translational reading frame
iv) splice site – create or destroy. A genetic mutation that inserts, deletes or changes a number of nucleotides in the specific site at which splicing takes place during the processing of precursor messenger RNA into mature messenger RNA.
v) Regulatory (promoter)
vi) Synonymous changes – no change to protein but change in binding sites for enhancer/suppressor proteins.
b) deletion/insertion
c) Dynamic mutations – repetitive. Increase or decrease in numbers of repeats within a repetitive sequence due to replication error.
- examples – huntingtons, Prader-Willi syndrome.
Nonsense and Frameshift mutations result in:
- the production of a truncated protein
- OR mRNA that is subject to nonsense mediated decay – no protein is ever actually produced
81
What are the disease-causing epigenetic changes?
- Deviation of epigenetic control – altered gene expression – altered cell function – disease
- Inherited disorders – Rett Syndrome, Fragile X syndrome.
- Disorders of imprinting – defects in methylation or centers controlling methylation, Prader-Willi and Beckwith Wiedemann.
82
Explain the effects of mutation on normal cell function
Change at sequence level leads to:
a) Altered transcription and RNA processing
b) Altered translation
c) Altered post-translational modification and folding
These in turn effect mature protein:
- amount of protein produced
- subcellular localisation
- biological function
- degradation/accumulation
- interaction with other subunits/cofactors
Outcome is either:
a) LOSS of function
- protein has reduced or no function, or,
b) GAIN of function - protein has new abnormal function
83
Discuss gain of function mutations
- often dominant
- often missense
- overexpression of a protein, constitutive activation of a receptor of open conformation of a channel, aggregation of a protein.
84
Discuss loss of function mutations
- often recessive
- product has reduced or no function
- can be missense, nonsense, Frameshift or splicing variants
EG – CYSTIC FIBROSIS:
- can have mild to classic (severe) disease
- loss of function mutations in CFTR gene which encodes chloride channel. Defective protein folding results in destruction of protein in ER and reduced expression of the CFTR protein at the cell surface.
- impaired transport of chloride ions and water across cell membrane results in thickening of secretions in lungs, pancreas and other organs.
- recessive
85
What is the nomenclature of genome variants?
- c. denotes change at DNA coding level
- p. denotes change at amino acid or protein level
- [=] means heterozygous so change isn’t on other gene. You will see two colored peaks on sanger sequence
- [0] means other chromosome is missing
- * means nonsense mutation so resulting codon is a stop codon.
