Week 3 Part 1 Flashcards
1
Q
What is the definition of a genome?
A
All the genetic material in the chromosome of a particular organisms
DNA in all cellular life form, RNA in some viruses
2
Q
Define Genomics
A
The study of genes and their function
3
Q
What does the nuclear genome contain and divided into?
A
Contain - approx 3,200,000,000 nucleotides to form 24 chromosomes
Divided into 24 linear molecules
4
Q
What is mitochondrial genome?
A
Circular double-stranded DNA molecule
Contain 16,569 nucleotides
5
Q
What does the mitochondrial genome give rise to?
A
- 37 genes coding for 2 rRNAs (12S abs 16S)
- 22 tRNAs
- 13 polypeptides
It is a maternal origin
6
Q
What is mitochondria?
A
Central role in cellular energy provision
Organelles Contain their own genome with modified genetic code
Not self supporting entities
Rely heavily for their function on imported nuclear gene products
7
Q
Where is the mammalian mitochondrial genome transmitted?
A
Exclusively though the female germ line
8
Q
What are mt-DNA encoded polypeptides?
A
All subunits of the enzymes complexes of the oxidative-phosphorylation system
9
Q
What is a genomic biomarkers?
A
A measurable DNA and/or RNA characteristic that is an indicator of normal biological process, pathogenic process, and/or response to therapeutic or other intervention
10
Q
What is the definition of genomic biomarkers used by?
A
- European Medicines Agency (EMA)
2. Food and Drug Administration (FDA)
11
Q
What can a genomic biomarker be a measurement of?
A
- The expression of a gene
- The function of a gene
- The regulation of a gene
12
Q
What can a genomic biomarker consist of?
A
One or more deoxyribonucleic acid (DNA)
And/or ribonuclease acid (RNA) characteristics
13
Q
What do DNA characteristics include?
A
- Single nucleotide polymorphism (SNP)
- Variability of short sequence repeats
- Haplotypes
- DNA modifications e.g. methylation
- Deletion or insertion of a nucleotide
- Copy number variations
- Cytogenetic rearrangements e.g. translocations, duplications, deletions or inversions
14
Q
What do RNA characteristics include?
A
- RNA sequences
- RNA expression levels
- RNA professing e.g. splicing and editing
- MicroRNA levels
15
Q
What doesn’t the definition of a genomic biomarker include?
A
Measurement and characterisation of protein or low molecular weight metabolites
16
Q
What do GWAS use?
A
Whole genome sequencing
17
Q
What is whole genome sequencing?
A
The entire genome being broken up into pieces of 2000, 10,000 and 50,000 base pairs long
18
Q
What does a huge percentage of the human genome consist of?
A
Repeated non-coding sequences dispersed throughout the genome
Include:
- LINES
- Minisatellites
Multiple copies of the same sequence that does not appear to have any valid function
19
Q
Whole exome sequencing
A
32 million bases
Collect blood Extract and fragment DNA Capture exome DNA with probes Recover only exome DNA fragments Sequence on next generation platform
20
Q
What is single nucleotide polymorphism?
A
- Altered single nucleotide (e.g. A,T,C,G) in the genome
- SNP are found in coding and predominantly in non-coding regions
- SNP occur with a very high frequency, hence most common form of genetic variation
- Identify whether a SNP occurs more often in people with a disease than controls
- SNP do not cause disease, but affect chance of developing disease, response to pathogen, drug etc
21
Q
When do you get Synonymous (silent mutation) of SNP?
A
If SNP lead to same polypeptide sequence
22
Q
When do you get non-synonymous (missense & nonsense mutation)
A
If SNP lead to different polypeptide sequence
23
Q
Who suggested that ~50% of a persons risk of becoming an addict is due to genome?
A
Family studies including monozygotic twin, dizygotic twin, adopters and siblings
24
Q
Who typically smokes more and thus have a greater risk for smoking-induced diseases?
A
Individuals with fast nicotine metabolism
25
What is dependent on the rate of nicotine metabolism?
The efficacy of smoking cessation pharmacotherapy
26
What was the objective of GWAS of a biomarker of nicotine metabolism?
Use nicotine metabolite ratio (NMR) in GWAS to identify novel genetic variants influencing nicotine metabolism
27
What was the Heritability estimates obtained for NMR using monozygotic and dizygotic twins of the Finn Twin cohort?
0.81
28
Individuals with fast nicotine metabolism
1. Smoke more (finance and health implications)
2. Have a greater risk of smoke-related disease
3. Poor response to smoking cessation pharmacotherapy
29
What is a predominant metabolite of nicotine with half life of 15-20 hours?
Continine
30
Why do you use nicotine ratio?
To measure:
1. Nicotine metabolism rate
2. Total nicotine clearance
3. CYP2A6 activity
31
What is the equation of NMR?
3-hydroxycotinine/ cotinine
32
How many smokers were identified from serum samples using liquid chromatography/ tandem mass spectrometry?
1518
33
What are some of the various demographic and hormonal factors that NMR is influenced by?
1. Ethnicity
2. Sex
3. Hormone replacement therapy
4. Use of oestrogen containing contraceptive pills
5. Body mass index
6. Alcohol consumption
7. Cigarette consumption
34
What was the method for study for GWAS of a biomarker of Nicotine metabolism
1. Written informed consent
2. GWAS used to identify novel genetic variants influencing nicotine metabolism in whole blood DNA
3. Compare data against 1000 Genome project data looking for SNP
4. FinnTwin12 is a population-based longitudinal study of 5 consecutive birth cohort, examine genetic and environmental determinate of health-related behaviours
35
What was the results of GWAS of a biomarker in nicotine metabolism?
1. At the region 19q13.2, 719 SNPs were identified within CYP2A6 gene
2. Strongest association was for CYP2A6 in Intron 4
3. At the region 19q13, SNPs were also identified within CYP2B6, CYP2A7 EGLN2 and NUMBL genes
36
What was the Nicotine Addiction (Discussion)
1. Study aim was to identify novel genetic variants affecting nicotine metabolism
2. Several members of cytochrome P450 gene family (CYP2A6, CYP2B6, CYP2A7, CYP2F1) involved
3. Top SNP, rs56113850, was located in intron 4 of CYP2A6 accounts for 14-23%
4. 2nd SNP, rs113388603, was located in 5.9kb upstream of CYP2A6 accounts for < 1% in NMR
37
What are the reasons for the SNP involvement in NMR does not account for 81% heritability based on the twins modelling?
1. Lack of CYP2A6 coverage on duplications, translocations and rare variants
2. High sequence Homology between between CYP2A6, CYP2A7 and CYP2A13 prohibit variant detection
3. Analysis can not detect rare SNP that have low effect size
4. Relatively small sample size
38
Genomic Mitochondrisl DNA biomarker?
1. Mitochondria are currently believed to play important role in the neurodysfunction and neurodegeneration that underlie PD
2. Mitochondrial dysfunction has been considered a common feature of PD
39
When did the fields interest in mitochondria begin?
1. Mid 1980s
After Parkinsonism-inducing toxin identified by Langston et al
Toxin: N-methyl-4-phenyl-1,2,3-tetrahydrolyridine (MPTP)
40
What does MPTP inhibit?
Complex 1 in the MPP+ form
41
What has the ability to prefentially kill dopaminergic neurons of substantia nigra?
Rotenone
| Spares the integrity of other cell types
42
What does evidence of MPTP indicate?
- after astrocytes take up MPTP and convert it to MPP+
- release it to the extracellular space
- neuron dopamine transporter prefentially absorb the toxin
43
What is Parkinson’s disease?
```
Age-related neurodegenerative disease
Temor
Rigidity
Bradykinesia
Speech issues
Positive response to L-dopa treatment
```
44
What did MPTP cause?
Parkinsonism symptoms in drug users
45
What is MPTP?
Inhibitor of complex 1 of electron transfer chain in mitochondria
46
What resulted in a vicious circle of oxidative stress and bioenergic failure?
Complex 1 mediated reactive oxygen species formation link with PD
47
What can PD-associated genes (e.g. PINK1, Parkin) affect?
Mitochondrial structure
Function
And turnover
48
What is reduced Parkinson DNA copy number a biomarker for?
Aetiology of PD
49
What are the methods of mtDNA quantification?
Asses the copy number of mtDNA in
n= 363
Peripheral blood samples
n=151 substantia nigra pars compacta tissue samples
n= 120
Frontal complex tissue samples from community-based PD
50
What was the method for biomarker of Parkinson’s disease ?
1. Total DNA was extracted from blood and tissue samples using standard methods
2. Quantification of mtDNA was performed in triplicate by multiplex Taqman qPCR
3. Application of the mitochondrial genes MTND1 and MTND4 and nuclear encoded genes B2M
4. Use serial dictions of cloned vectors to ensure reaction linearity and for standard curve quantification
51
What was the results of the study of biomarkers for Parkinson disease
Initially investigated the role of mtDNA copy number in peripheral white blood cells, identifying a significant reduction in mtDNA copy number in PD cases when compared to control
This association was dependent of age and gender
Circulating cell-free mtDNA is present in CSF
Analysis of two mtDNA targets: MTND1 and MTDN4 genes
Shows a decrease mean mtDNA copy number in
CSF for PD at start and end of study
Decreased mtDNA copy number in PWBC and SNpc but no change in frontal cortex
Significant association with decreased mtDNA copy number in PWBC and smoking
52
What is the discussion of biomarkers for Parkinson disease?
MtDNA copy number is important component in the pathoetiology of PD
Using different tissues - demonstrated that in high-turnover tissues such as PBC and in vulnerable post-morgen tissues such as SNpc, mtDNA copy number is reduced and may be an important early biomarker for PD onset
There is a PBC copy number reduction in Huntington disease and a reduction of mtDNA in pyramidal neurons in Alzheimer’s disease hippocampi
53
What does a decreased mtDNA copy number suggest?
Mitochondrial dysfunction
54
What is Genomic Mitochondrial DNA biomarker (limitations)?
The role of decreased ccf-mtDNA in PD is unknown
Cancer had increased ccs-mtDNA level suggested to coorelate with increase apoptosis
Intensity care unit mortality is correlated with increased ccf-mtDNA levels
55
What is epigenetics?
The study of changes in organism caused by modification of gene expression without changing the DNA sequence
56
What does epigenetics switch on and off?
On - genes only for neuronal features (e.g. produce dendrites, axons)
Off - genes for non- neuronal features (e.g.. produce insulin, melatonin)
57
Where does epigenetics signals come from?
```
Within cell (e.g. during division)
Neighbouring cell (e.g. cortical layers in cortex)
Environment (temperature, diet)
```
58
What are the features of epigenetics?
1. Epigenetics control genes
2. Epigenetics is everywhere
3. Epigenetics makes us unique
4. Epigenetics is reversible
59
Define Polyphenism
When two or more different phenotypes are produced from a single genotype in response to the environment
60
What does polyphenism provide?
Fascinating opportunity to study how the environment affects an animals genome and how this produces changes
61
What is a best-known case of polyphenism?
Caste development in honeybees
62
What do honeybees have?
Two female castes:
1. Workers
2. Queens
63
What do honeybees differ in?
Behaviour and physiology
| As a result of their larval diet
64
What are larvae destined to become queens fed?
Royal jelly
A nutrient rich diet
65
What are worker larvae Fed?
A more dilute diet known as worker jelly
66
What does the difference in larval nutrition set up?
Different developmental trajectories they are characterised by change sin gene expression throughout larval development
67
What are polyphenism often associated with?
Caste and are found in many insects
| Bees, ants and beetles
68
What is DNA methylation?
Epigenetic mechanism that occurs by the addition of a methyl group to DNA
Modify the function of genes and affecting gene expression
69
What is the most widely characterised DNA methylation process?
Covalent addition of methyl group at the 5-carbon of the cytosine ring resulting in 5-methylcytosine (5-mC)
Methyl groups project into major groove of DNA and inhibit transcription
70
What is the function of DNA methyltransferase?
The addition of methyl groups is controlled at several different levels in cells
71
What 3 DNMTs are required for the establishment and maintenance of DNA methylation patterns?
DNMT1
DNMT3a
DNMT3b
72
What does methylation at specific promoter region of a gene inhibit?
Gene expression
73
DNA methylation biomarker
Bisphenol A is an estrogenic endocrine disruptive widely used in the production of plastics
74
What affects sexual differentiation and behaviour?
Utero BPA exposure
75
What was hypothesised about BPA?
Disrupts epigenetic programming of gene expression in the brain
76
Where is BPA detected?
Urine of >90% examined US population
77
Bisphenol A
1. Estrogenic endocrine disruptor
2. Accumulate foetal and infant tissues
3. Disrupts normal neurodevelopment
78
DNA methylation biomarker (methods)
1. All procedures were performed with approval of institutional animal care and use committee at Columbia Univeristy
2. Pregnant mouse dams treated orally with either bisphenol A or vehicle (corn oil)
3. RNA and DNA extracted from P28 offspring brain tissue (cortical, hypothalamic, hippocampal)
4. RNA used to study gene expression via qRT-PCR on estrogen receptor 1 (Esr1), ESR2 and Estrogenic-related receptor gamma (Esrrg) genes
79
DNA methylation biomarker (methods)
1. DNA extraction
| 2. Bisulfite conversion of DNA samples (500ng)
80
What does sodium bisulfite convert?
All un-methylated cytosine to uracil, then following PCR only C remain are the methylated ones
81
How was CPG methylation assessed?
Using DNA sequencing (pyrosequencing) on ESR1, ESR2 and Esrrg1
82
What was the results for DNA methylation biomarker?
Examined the effects of prenatal BPA treatment on expression of genes essential for sexual differentiation of brain and behaviour
In prefrontal cortex, BPA treatment led to a marginal quadratic change in ESR2 expression in both sexes
In the hippocampus, BPA treatment led to no change in ESR1 expression
In the hypothalamus, BPA treatment led to sex-specific quadratic changes in ESR1, ESR2 and Esrrg expression
Low dose of BPA led to a loss or reversal of sex differences in ER-related gene expression evident in vehicle-treated offspring
83
What are Histone?
Proteins which stably interact with DNA to form nucleosomes and package DNA into chromatin
84
Why does Histones unwound?
Allow for gene transcription
85
Histone modification (intro)
Glioblastoma is the most common and lethal primary malignant brain tumour
Classified Into:
- astrocytic
- ependymal
- oligodendroglial
- choroid plexus tumours
86
What are the different grades for glioblastoma?
Low grade
- Grades 1 and 2: slow growing
High grade
- Grades 3 and 4: fast growing
Determines the type of treatment provided
87
Why study Histone modification?
Help understand the types of glioblastoma
88
Histone modification biomarker (methods)
1. Research conducted was approved by the Research Ethics committee of the Univerdity Hospital
2. In accordance to the Helsinki Declaration
3. Tumour samples from 43 patients were evaluated
4. Normal cerebral tissue from 11 patients were obtained
5. Extract total cellular RNA, then RNA reverse transcribed to single stranded CDNA
6. Use taqman qRT-PCR to identify HDAC using B-glucoronidase (GUS B) gene as endogenous control
89
High Histone deacetylase level
Decrease transcription
90
High Histone acetyltransferase levels
Increase transcription
91
What was the results for Histone modification biomarker?
MRNA levels of class II and IV HDAC were downregulated in glioblastoma compared to low grade astrocytomas and normal brain tissue
The protein levels of class II HDAC9 are lower in high-grade astrocytomas
Histone H3 (but not Histone H4) was more acetylated in glioblastomas than normal brain tissue
92
What is the discussion of Histone modification biomarker?
Negative correlation between HDAC gene expression and the glioma grade suggesting that class II and IV HDAC May play important role in glioma malignancy
Histone H3 is more acetylated in glioblastomas than normal brain tissue confirming the down regulation of HDAC mRNA in glioblastomas
93
What is clinically used to treat epilepsy?
Valproic acid/ valproate
94
What is a HDAC inhibitor?
Bipolar mania
95
What can be identified using genomic biomarkers?
Changes due to ageing and diseases
96
What can drugs that affect DNA methylation and Histone modification become?
Drugs of the future
| For treatment in neurological disease or aging
97
What can inhibit DNA methylation?
e. g. hydralazine (treat hypertension)
| e. g. valproate (treat epilepsy)
