Human Molecular Genetics Flashcards
1
Q
Why was the human genome sequenced? (5)
A
to:
- identify all human genes and their roles
- analyse genetic variation between humans as a species
- sequence the genomes of several model organisms used in genetics, such as the fruit fly
- develop new sequencing techniques and computational analysis
- to share genome information with scientists and the general public as fast as possible
2
Q
What is a genome?
A
a complete set of DNA of an organism
3
Q
What is the human reference genome?
A
A genome made from 10 anonymous volunteers that is used as a reference to compare individuals genome to
4
Q
What are some key findings from the human genome? (6)
A
- there are fewer genes than expected (we have about the same as a type of worm)
- less than 2% of our genome codes for proteins
- the genome is dynamic (contains evidence of recent evolution)
- we don’t know what many of our protein coding genes do
- most human genes are related to those of other animals
- all humans are 99.9% similar at sequence level
5
Q
Describe the makeup of the human genome:
- what percentage exons/coding?
- what percentage introns?
- what percentage regulatory sequence?
- roughly how many genes?
- about what percentage have an unknown function?
A
- 1.5% coding (exons)
- 20% introns
- 5% regulatory sequences
- ~21,000 genes
- about 25% still have an unknown function
6
Q
How do we find a gene?
A
- genes tend to start with ATG
- have open reading frame
- have exons and introns
7
Q
Which part of the genome is making functional proteins and what percentage of the genome does this make up?
A
the exons making up 1.5% of the genome
8
Q
What do the regulatory sequences do?
A
sit around the genes and determine whether the genes should be turned on or not
9
Q
How much of the human genome does introns, exons and regulatory sequence account for?
A
25%
10
Q
What are 4 different types of variation in the genome?
A
- SNPs
- STRs
- InDels
- CNVs (structural variants)
11
Q
What does SNPs stand for?
A
Single nucleotide polymorphisms
12
Q
What does STRs stand for?
A
Short Tandem Repeats
13
Q
What does InDels stand for?
A
insertions and deletions
14
Q
What does CNVs stand for?
A
Copy number variations
15
Q
What is the importance of variation?
- what is it the key driver for?
- knowledge of variation can be used to….? (4)
A
- variation is the key driver for evolution
- knowledge of variation can be used to diagnose genetic disease, to determine which drugs will work best on a patient, to determine our close relatives, or our species’ origin
16
Q
What are SNPs?
A
- common single base pair changes or variants
ie. sites in the DNA that vary within populations
17
Q
How common are SNPs?
A
- common: about 1 in every 300 nucleotides
18
Q
What are some effects of SNPs?
A
in the coding region: could change the protein (only if it changes an amino acid and only if it’s an important amino acid in the protein)
19
Q
What are STRs?
A
repeats of 2-5 nucleotides found in specific regions of the genome
20
Q
Give an example of an STR
A
Each person inherits 2 alleles, one from each parents which could vary in length
eg. at one site:
CAGCAGCAGCAGCAGCAGCAGCAG 8 repeats of ‘CAG’ from mum
CAGCAGCAG 3 repeats of ‘CAG’ from dad
this person is 3,8 at STR1
21
Q
What are InDels?
A
small insertions or deletions which causes a “frame shift” in the way DNA is read if in a protein coding region
22
Q
What are copy number variants?
A
A type of structural variation where individuals vary in the number of copies of a region of DNA they have in their genome
23
Q
How many CNVs do humans have?
A
10,000 found within and between genes
24
Q
What can we see from comparing genomes?
A
We can see what is the same and what is different and learn about which parts of the genome do what
25
How do we compare sequences?
By lining them up next to each other and marking each point where sequences are the same.
26
What is it called when DNA sequences are lined up next to each other and each point where sequences are the same is marked?
aligning
27
Differences in genomes of individuals with the same species might be associated with
- disease
- characteristics of an individual
- evolutionary history
28
Comparing genomes within a species can help us identify
variants that might be related to phenotype
29
What was found from the Denisova cave?
- 4 billion Neanderthal nucleotides
30
What were some findings when human DNA was compared to Neanderthal DNA?
- some of us carry Neanderthal DNA
- modern humans from Europe and Asia carry Neanderthal alleles
- those from Africa show no signs of these alleles
31
What do the findings when DNA between humans and Neanderthals were compared suggest?
That where modern human humans met Neanderthals, they interbred
32
What percentage of the genome of non-Africans is made up of variants that arose in Neanderthals?
2-4%
33
Neanderthal DNA adds to
the variation in our genome that might be related to our phenotype
34
What percentage of the genome of modern day Melanesians is made up of variants that arose in Denisovans?
4-6%
35
What does the presence of Denisovan alleles in our genome suggest?
Homo sapiens mated with Denisovans (as well as mating with Neanderthals)
36
Comparing genomes within a species can help us identify
variants that might be related to the phenotype
37
Comparing genomes with another species can help us
identify variants that might be related to the biology or an organism
38
What are mutations?
Permanent changes to the DNA sequence
39
Mutations can be (2)
inherited or acquired
40
Mutations that are inherited are called _______ and are passed by the _______
germ-line mutations
| gametes
41
Mutations can be acquired by somatic cells if
DNA gets damaged or is copied incorrectly
42
Can somatic mutations be passed onto the next generation?
no
43
Mutations are the driving force of
evolution
44
Mutations can be (3)
beneficial
harmfull
have no effect
45
The vast majority of mutations have
no effect
46
What does the outcome of the mutation depend on
environmental effects
| other genes
47
What are some environmental effects that the outcome of a mutation depends on?
diet
| exposure to toxins
48
Mutations can be _______ or __________ or give a
| _________ or ________
dominant
recessive
loss of function
gain of function
49
A dominant mutation is one that causes the phenotype when
Heterozygous
| only 1 copy of the mutant allele present
50
A recessive mutation is one that causes the phenotype when
Homozygous
| 2 copies of the mutant allele need to be present
51
What is a loss of function mutation?
A mutation that might break a gene
52
Loss of function mutations are often ______ (and why)
recessive because a normal copy of the gene exists on the other chromosome which can replace the lost function
53
What is a gain of function mutation?
When a mutation causes the gene to work too well, or to do something unexpected
54
Gain of function mutations are often _______ (and why)
dominant because having an allele that works too well will not be replaced by the other gene
55
What is a monogenetic disease?
a disease caused by one gene
56
What are some monogenetic diseases?
- haemophilia
- Huntington's disease
- Cystic fibrosis
57
Describe haemophilia
- blood clotting disorder
- when you cut yourself, your blood starts to clot
- this is a very finely balanced chemical reaction involving a bunch of proteins that detect when blood is flowing abnormally and you start forming that clot structure.
- haemophilia A results from impairment or absence of clotting factor VIII
- haemophilia B results from impairment or absence of clotting factor XI
58
Describe the inheritance of haemophilia
- caused by an inversion mutation on the X chromosome
| - mutations are loss of function
59
What is an inversion mutation?
A piece of DNA has popped out, turned around and gone back into the sequence. This scrambles the haemophilia protein so its doesn't work anymore
60
Because haemophilia affects the X chromosome, are males or females more likely to inherit the mutation? What are the chances?
- Males are more likely to have haemophilia
- Women have two X chromosomes and are rarely affected
- Sons of women who are carriers have a 50% chance of inheriting the disease
61
Describe Huntington's disease
- neurodegenerative disease
- progressive tremors
- involuntary movement
- onset in midlife (30-50)
- no effective treatment
62
Describe the inheritance of Huntington's disease
- autosomal dominant inheritance
63
Describe the genetic cause of Huntington's disease
- mutation of HTT gene in chromosome 4
- caused by the expansion of CAG triplet repeat in the HTT gene
- In HTT, there is a patch of CAG repeats and CAG encodes glutamine in the protein
- the protein therefore has a long polyglutamine tract
- the protein become unstable and it fragments, clumping together in nerve cells and damaging them
64
How can you test for Huntington's disease?
- putting a primer at each end of the CAG region
- do PCR to determine how far apart the primers are which can tell you how long the CAG region is
- the length of the CAG region can affect the chances of getting Huntington's
65
How many CAG triplets affect the chances of getting Huntington's disease?
10-35 copies: normal
| 40+ copies: disease develops
66
What are some symptoms of Cystic fibrosis? (4)
- lung infections
- pancreatic insufficiency
- congenital absence of vas diferens in males
- salty tasting skin
67
The inheritance of Cystic fibrosis is
autosomal recessive
68
What is the mutation that causes Cystic fibrosis?
- three base pair deletion in the CTFR gene
- the protein is abnormally processed and degraded because it goes to the wrong part of the cell
- this causes thickening of cells secretions
69
How can you find potential disease genes?
1. sequence the genome
2. compare this with the human reference genome and determine what is different
3. eliminate the variants in common (because they are not causing the disease)
4. look at the different variants
5. eliminate the ones that are not predicted to be harmful
6. validate and test the variants that are predicted to be harmful
70
What are polygenetic diseases?
- diseases that involve many genes acting together or environmental factors interacting with genes
71
What are some examples of polygenetic diseases?
```
obesity
diabetes
gout
bipolar disorder
rheumatoid arthritis
```
72
How can you find a polygenetic disease?
1. compare the genomes of cases (ie. people with the disease) with a control (people without the disease)
2. identify variations between the two groups
3. of these variations, determine the shared variations within the cases group
4. validate and test these variants
73
Most genetic disorders are ________ not _________
probabilistic not deterministic
74
What does "most genetic disorders are probabilistic not deterministic" mean?
it means that for most genes, having disease related variation does not mean you will get the disease because the diseases come about through a combination of variants and the environment
75
Where gene change alters the phenotype, it gives a clue to the
gene function
76
If we can find or create mutants in the related genes in other animals, we may learn
what the functions of those genes are in humans
77
How do we use genetic techniques to find out what a gene does?
Using functional molecular genetics
78
Describe the process of functional molecular genetics
1. study organisms that are naturally occurring for that gene
2. increase the rate of mutation by intensive breeding or chemicals
3. select for a phenotype of interest and sequence the genome to identify the mutation (genetic screen)
4. once you have a gene you are interested in, you can insert it into another organism using transgenesis or genetic engineering
5. you can also break a particular gene to see what happens using targeted mutation, gene knockout or reverse genetics
79
What are some model organisms can be used to make mutants?
Mice
Zebrafish
Drosophila
80
Why can some organisms be used to make mutants?
Because we share many of our genes with them and because these model organisms can easily raised in a controlled environment and are easy to manipulate genetically
81
Mutants can be made by treatment of gametes with mutagens such as
X - rays
| Chemicals
82
What is the purpose of transgenesis?
To understand how genes work / what a gene does.
To engineer useful protein products (synthetic biology).
For gene therapy approaches.
To study the effect of making a foreign protein or of making a protein in the wrong cells.
83
What is transgenesis?
Where we add DNA from a different organism to a genome to make a new protein or to replace a defective gene
84
What is gene therapy?
Delivery of a good copy of the gene to sick cells so they have a good copy of the gene to make a good protein from
85
What do you need for trangenesis?
regulatory sequence and the coding part of the gene that you want
86
What is the purpose of the regulatory sequence?
A regulatory sequence is something to make the gene work in the organism you are putting it in because it controls how that gene is deployed (moved into action)
87
Give an example of trangenesis and explain how it works
Putting the fluorescent jellyfish gene into mice
You can inject a mouse embryo after fertilisation and inject the DNA that you want. The DNA will therefore become part of the DNA of the mouse
88
Give examples of how transgenesis can help humans
- Factor IX for haemophilia B in hamster cells
- spider silk produced by transgenic goats, in their milk
- human insulin produced by bacteria for treatment of type 1 diabetes
- Brewer's yeast modified to produce cannabinoids
89
How can you find out whether a variant is pathogenic?
1. Sequence the genome
2. Map and compare it to the human reference
3. Rule out the common variants
4. Of the novel variants, rule out those predicted to be benign
5. Of the variants that are predicted to be harmful, validate and test them
90
How can we test whether a variant gene is pathogenic?
- we can damage, or modify, the gene we are interested in by genetically modifying an organism or cell line
- by examining the organism, we should be able to work out what that gene normally does
91
What is one method of modifying/damaging the gene?
CRISPR - Cas9
92
What is Cas-9
associated protein 9
93
What is Cas9 and what is its purpose?
It is a nuclease that cuts double stranded DNA molecules
94
Explain how CRISPR-Cas9 works
- Cas9 protein contrains active sites that can cut DNA
- a guide RNA binds with the Cas9 protein forming a complex that is allowed to then be introduced into the cell
- In the nucleus, the complementary sequence of the RNA binds to part of the target gene
- the active site of the Cas 9 protein cut the DNA on both strands
- this results in a cut in the target gene
- the broken strands of DNA are "repaired" by the the cell in two different ways
95
What is the purpose of the guide RNA?
It guides the Cas9 protein to a target gene that only binds to the gene of interest
96
Describe the structure of the RNA that is used in CRIPSR-Cas9?
There is a complementary sequence that can bind to the target gene
The RNA also binds to itself in a structure called a hairpin which makes the RNA stable and it also means that it can fit nicely into the cas-9 enzyme.
97
What are the two different ways in which the cell "repairs" the broken DNA after Cas9 broke the strands?
1. no template is provided and repair enzymes insert and/or delete random nucleotides (InDels) making the gene non-functional or mutated
2. if a repair template is provided, it is possible to use this to "edit" the DNA sequence at the cut site ("gene editing")
98
What are the two types of genetic disease?
Somatic diseases
| Germline diseases
99
Describe a somatic disease
- the target cells or organs are affected
- does not affect the next generation
- often single gene diseases caused by loss of function mutations
100
What are some methods of fixing somatic diseases?
Gene therapy
| CRISPR-Cas9 is also being explored as an example
101
What's an example of a somatic disease and how can it be fixed?
- Cystic fibrosis
- caused by mutations that alter the function of the Cl- ion channel encoded by the CFTR gene
- gene therapy can be used to fix it
102
Describe how gene therapy can be used to help Cystic fibrosis
Delivering DNA with a functional copy of the CFTR gene to lung epithelial cells via nebuliser (drug delivery device used to administer medication in the form of a mist inhaled into the lungs). The extra copy of the DNA makes good CFTR protein, restoring function into some cells.
103
What are some ways to fix germline diseases?
- preimplantation genetic diagnosis
- three parent babies
- CRISPR gene "edited" babies
104
Describe preimplantation genetic diagnosis
IVF can be used to make embryos from the parents egg and sperm. These embryos can be tested before implantation, and only healthy embryos implanted
105
Describe three parent babies
Important genes are found in the mitochondria which is inherited from the mother. If these are faulty, a donor egg with good mitochondria can be used. Therefore the baby had DNA from 3 parents: sperm from the dad, nucleus from the egg from the mum, and the mitochondria from the egg of another women
106
What is the purpose of CRISPR-Cas9?
It is one method to break or modify a gene replicate a possible disease causing variant in a model organism, to see if the disease develops as a result
107
What does totipotent mean?
The cells have the potential to make any cell in the whole body including the placenta and embryo
108
What does pluripotent mean?
The cells that have the potential to make any cell in the whole body but not the placenta and embryo
109
What are the stages of embryo development?
1. an 8 cell stage
2. a cell polarisation
3. compaction
4. the inner, apolar cells are cut off
5. forms a blastocyst
110
Describe cell polarisation
There is an inside and an outside forming. The outside makes microvilli and nucleus moves towards inside
111
Describe compaction
Cells are tightly stuck together
112
Describe the process where the inner, apolar cells are cut off
The 8 cells divide in the plane which means that the inner cells are cut off from the outside environment (intrauterine environment)
This makes them do completely different things
113
Cells become more __________ and less __________ during embryo development, except for ______ _______ and ________ __________ (______ and _______)
specialised
flexible
stem cells
germ cells (egg and sperm)
114
Describe the blastocyst
There is the inner cell mass (ICM) and the trophectoderm
115
What is the ICM?
Is it the inner cell mass that is formed from the division of apolar cells that have been cut off
These continue to divide to form you
116
What is the trophectoderm?
The embryos contribution to the placenta.
117
What is a stem cell?
A relatively unspecialised cell that can both reproduce itself indefinitely and, under appropriate conditions, differentiate into specialised cells of one or more types
118
Summarise why the human genome was sequenced
To find all the human genes and to identify the types and extent of variation in the human population
119
Knowledge of variation can be used to (4)
1. diagnose genetic disease
2. determine which drugs will work best in a patient
3. determine out closest relatives
4. determine out species origins
120
The embryo begins as a small number of naïve, __________ cells
totipotent
121
Embryonic stem cells can give rise to all cell types except
trophectoderm
122
Embryonic stem cells are not
differentiated
123
Embryonic stem cells are an example of
pluripotent cells
124
What are pluripotent cells?
Cells that are capable of giving rise to many different cell types
125
What is cell differentiation?
When cells become specialised in structure and function
126
Define totipotent, pluripotent and multipotent
Totipotent:
such as a fertilised egg which is able to give rise to all cell types via cell division (including the placenta)
Pluripotent:
such as embryonic stem cells can become any cells of the human body
Multipotent:
stem cells that give rise to both stem cells and cells that will differentiate into one or more (but not all) types of functional tissue cells. Examples include adult stem cells such as those from blood or the bone marrow
127
Describe genomic equivalence
differentiated cells contain all the DNA required to build an entirely new organism
128
Embryonic stem cells are harvested from the
inner cell mass
129
Umbilical cord stem cells are _________ as they are _________ _________ _______ __________
multipotent
| immature blood stem cells
130
What are haemotopoietic stem cells?
Blood stem cells that are found in the bone marrow and can be used for transplants
131
What is gene editing?
A type of genetic engineering in which genomic DNA is altered, removed or replaced
132
What is an iPSC?
Induced Pluripotent Stem Cell: a laboratory technique that "deprogrammes" differentiated cells through manipulation, restoring them to a pluripoent state
133
What is a stem cell?
Any relatively unspecialised cell that can produce, during a single division, two identical daughter cells or two more specialised daughter cells that can undergo further differentiation, or one cell of each type
