Using Molecular Genetic Methods Flashcards
Topic 1
DNA variation and Human Disease
What is a typical cell made up of?
and
where is genetic information stored?
the typical cell is made up of the plasma membrane on the outside, followed by the cytoplasm, and then in the centre, you have the cell nucleus.
Genetic information is stored in the nucleus in the form of chromosomes
How many pairs of chromosomes do humans have?
23
So, how many make up the human genome?
46 chromosomes
One of each of these pairs comes from your mother, and the other comes from your father. And each of these chromosomes contains highly coiled and condensed DNA.
True or False?
TRUE
DNA stands for?
Deoxyribonucleic Acid
What are DNA molecules?
double-stranded helices located in the nucleus
What cells are the only cells without DNA?
Red blood cells
DNA code is centred on the sequence of base pairs along the length of the DNA double helix, and there are four types of bases. What are they?
1) Adenine
2) Thymine
3) Guanine
4) Cytosine
A base on one strand of the double helix strictly pairs up with another base on the other strand. What is the pairing?
A goes with T
G goes with C
How many base pairs do we have in or genome roughly?
6 billion
What is half a set of chromosomes (23) called?
Haploid
What is a full set of chromosomes (46) called?
Diploid
All genetic differences between people come from only what % of difference?
0.1% difference in the base pairs along their DNA
How big is DNA?
DNA is incredibly long
Base pair: 0.34nm
Diploid cell: 2m of DNA
74 trillion metres of DNA in total.
This would stretch from here to the Sun and way beyond
emphasise how massive DNA is, and how much information it contains, and just how spectacular your body is in making it so compact.
is DNA negatively or positively charged?
DNA is negatively charged
allows it to wrap itself around positively charged what?
Histones
it’s the tight coiling of DNA which helps to keep it so compact.
The structure which is formed when DNA molecules wrap themselves around histones is referred to what?
nucleosomes
What is the purpose of nucleosomes?
Nucleosomes are folded and tightly coiled to form fibres that make up the structure of the chromosomes
Each Nucleosomes consists of how many histone proteins?
8
Chromosomes have a very distinctive structure. They have a long Q arm and a shorter P arm held together by a structure called a what?
Centromere
What is a karyotype?
A karyotype is an individual’s complete set of chromosomes
If there is the presence of a Y chromosome in the karyotype this tells us this karyotype is what gender?
MALE
The X and Y chromosomes are referred to as?
Sex chromosomes
all of the other 22 pairs of chromosomes are referred to as
autosomal chromosomes
What is a gene?
• the basic unit of heredity
• short sections of DNA that contain
instructions to make proteins
• each person has two copies
What defines the structure of a gene and confer certain functional properties?
Nucleotide sequences
For instance, the promoter sequence in the 5’ flanking region of the gene binds what?
Transcription factors
And is vital in what?
is vital in the initiation of gene expression
A typical sequence at a promoter region includes repeats of?
T-A-T-A, thymine and adenine, often nicknamed a TATA box.
Other regions in the 5’ flanking region are also important in regulating the rate of gene expression.
A mutation is what?
a permanent change in the nucleotide sequence of the genome of an organism.
What are 3 common causes of DNA mutations?
- unrepaired damage caused by radiation or chemical mutagens
- errors in replication or recombination
- transposons (jumping genes)
Explain in more detail the mutations caused by unrepaired damage to DNA?
Mutations often arise from damage caused by radiation or chemical mutagens.
Long-term exposure to UV rays can damage DNA leading to skin cancer.
UV rays break hydrogen bonds between the two strands.
Covalent bonds can form between adjacent pyrimidine nucleotides.
Can the repair mechanism correct some errors of this mutation?
Repair mechanisms can correct some errors, but uncorrected errors lead to changes in the DNA code being passed on.
In cancer, DNA damage can lead to abnormal cells undergoing what?
mitosis at an alarming rate, and becoming immortal - the cells do not die as they should.
Explain in further detail the errors in DNA replication or recombination?
the swapping of DNA between chromosomes during egg or sperm production
Where there are large changes to genetic material, there is lots of room for error.
If the cell’s repair mechanisms don’t correct the erroneous sequences, a mutation occurs.
Explain in detail transposons mutations?
Transposons were discovered by Barbara McClintock in the 1940s.
She found mobile genetic elements in maize could insert themselves randomly into the genome.
McClintock noticed insertions, deletions, and translocations.
Cells contributing to each kernel had different mutations, causing neighbouring kernels to have characteristic colours.
What % of the maize genome is made up of transposons? ad are transposons present in human?
85% and yes
What are transposons also called?
Jumping genes
Mutations can occur in germline or somatic cells. Germline mutations (sperm or egg cells) occur in what type of cell?
Haploid cells
these mutations may be transmitted to the next generation producing an individual with the mutation. True or false?
TRUE
Somatic mutations are present where?
Diploid cells (all other cell types)
Can a mutation in somatic cells be carried on to the next generation?
NO
but it may affect the person carrying it
Mutations can be good, which we call advantageous, or they can be bad, which we call?
Deleterious
So a mutation remains within the population if it increases the reproductive fitness of an organism, and increases the chances of passing on their genes, and it disappears if it leads to disease states or early death
true or false?
TRUE
A mutated form of a gene that appears and remains in a population over a period of time becomes a stable new variant of the gene, known as a what?
Allele
What are the 2 types of chromosomal mutations?
1) Large scale mutations
such as a change in the whole chromosome number or a modification to a chunk of a chromosome
2) Small scale mutations on a nucleotide level
There are several types of chromosomal mutation. Some involve a change in the number of chromosomes, which we call what?
aneuploidy (change in number)
Other mutations involve changes to chromosome structure, which may involve what?
duplication, deletion, translocation, or inversions
What is meiosis?
Meiosis is the process in which haploid cells are formed from somatic diploid cells.
What is the process of meiosis? 6 steps
- DNA replication of the genetic material
- Homologous chromosomes of similar type
• Homologous chromosomes break and swap over, mixing the genetic material –
genetic recombination
- Meiosis I - genetic information is halved
- Meiosis 2 - genetic information is halved again
The result is haploid egg or sperm cells, 1⁄2 the chromosomes of body cells
Sperm and egg meet - formation of diploid zygote
What is aneuploidy?
Aneuploidy - a cell or organism contains a different number of chromosomes characteristic of its species
When does it occur?
Aneuploidy occurs during a malfunction in meiosis when the chromosomes do not separate properly.
example, instead of a haploid cell having one copy of the chromosome it has zero or two copies.
What is the most well known case of aneuploidy?
Down’s syndrome
What is trisomy 21
It involves the gain of a single extra chromosome, chromosome 21, which exists in three copies in all the person cells. So it’s called trisomy 21.
How is trisomy 21 characterised?
it’s characterised by a low IQ, folds over the eyes, short and broad hands, and below average height.
Klinefelter syndrome is another case of aneuploidy and involves the gain of what?
Giving rise to the karyotype?
Gain of an X chromosome
the female sex chromosome, and gives rise to the karyotype XXY
What is the number which Klinefelter’s occurs in, in the population?
1 in every 1,000 live births
How is Klinefelter characterised?
characterised by underdeveloped testes, some breast development, taller than average height and low IQ.
What is Turner’s syndrome?
Turner’s Syndrome involves loss of a single sex chromosome and inheritance of a single X
We refer to a loss of one of the pairs of chromosomes as?
Monosomy
Turner’s occurs in 1 in every?
10,000 female live births
99% die before birth
What are the symptoms for Turner’s syndrome?
The symptoms are shorter than average height, web-like necks, poorly developed breasts, immature internal organs, and reduced ability to interpret spatial relationships.
Structural mutations tend to occur due to what?
errors during meiosis
- they begin with one or more breaks in the chromosome.
- Wherever the break occurs, the broken ends remain exposed and vulnerable to degradation.
- Broken ends of chromosomes are sticky and can adhere to other broken ends.
Chromosome deletions involve the loss of what?
Genetic material.
Centromeres are not required for proper division of genetic material during mitosis and meiosis.
True or false?
FALSE
Centromeres are required for proper division of genetic material during mitosis and meiosis.
Removal of the centromeres by deletion will lead to what?
An aborted foetus
any chromosomal deletions present in the living individual will not include the centromere, but can involve almost any other part of the chromosome. True or false?
TRUE
An example of a deletion is Cri-du-chat syndrome, which is caused by what?
caused by deletion in the p arm of chromosome 5.
The syndrome gets its name from the characteristic cry of affected infants, which is similar to that of a meowing kitten.
This occurs due to problems with the larynx and nervous system.
Later in life, sufferers exhibit cognitive and behavioural problems.
Another deletion mutation causes Prader-Willi syndrome. This solution is in the what part?
q arm, chromosome 15
Prader-Willi syndrome results in what characteristics?
low muscle tone, short stature, incomplete sexual development, and cognitive disabilities.
Chromosome duplications
Chromosome duplications can arise when?
when chromosomes undergo crossover or genetic recombination and results in the doubling up of a segment of DNA.
And these duplications occur why?
It occurs when a surplus genetic region fails to break off the main body of one homologous chromosome.
So this genetic region is duplicated on the segment of chromosome, joining from the other homologous chromosome.
What is a disorder which is an example of a duplication?
Pallister-Killian syndrome is an extremely rare condition, in which the short arm of chromosome 12 is duplicated. It’s usually a mosaic chromosomal mutation
What is a mosaic chromosomal mutation?
an individual can carry some cells with and some cells without the mutation.
And in Pallister-Killian this results in what?
developmental disability and epilepsy, amongst other symptoms.
Explain Chromosome inversions?
and what chromosome are they commonly on?
- p and q arms bend in on themselves and cross over
- segment can break off and be reintegrated at 180 degrees
- may be phenotypic consequences when the breakpoints occur within genes
- most common tends to be on chromosome 9
Explain Translocations in more detail?
Including balanced and unbalanced
- involve swapping genetic information from different chromosomes
- a change in position of segment of a chromosome to a different location on a non-homologous chromosome
- balanced - material is just moved but functional
• unbalanced - exchange of material is unequal resulting in extra or
missing genes
Chromosomal mutations in psychiatry
Scale mutations can be a result of what 3 things?
Deletions
Insertions
point mutations
How do point mutations occur?
Usually occur during DNA replication
- Nucleotides bind to the exposed strands of DNA
- DNA polymerase catalyses the formation of bonds between adjacent nucleotides
- Sometimes incorrect nucleotide pairings can form, which can lead to a point mutation
What are the 2 types of point mutations which can occur?
transitions and transversions
What are transitions?
A transition point mutation is where a base is replaced by another base of the same type
Give me an example of a transition point mutation?
a two- ringed purine, adenine, is replaced by guanine,
or
where a single-ringed pyrimidine base is replaced with another pyrimidine base– so cytosine to thymine, or vice versa.
What are the 2 ringed purines?
adenine and guanine
What are transversions?
when a pyrimidine is replaced with a purine, such as cytosine to guanine
or
adenine to cytosine
What are the 2 pyramidines?
Cytosine and thymine
Point mutations can be of four types, each with different effects
What are the 4 types?
missense
nonsense
neutral
silent mutations
Explain Missense mutations?
a base pair change causing a change in the messenger RNA codon
Change in mRNA - different amino acid is inserted into the polypeptide.
If sequence changes, it might attract a different tRNA and amino acid, leading to different amino acid and polypeptide.
What is a codon?
- a codon is a three-nucleotide sequence along the messenger RNA molecule
- which attracts complementary transfer RNA coupled to a specific amino acid.
What are nonsense mutations?
- nonsense mutation are base-pair changes
- causing a change in the messenger RNA codon to a stop codon
- terminating the polypeptide chain
- to form a truncated protein.
What are neutral mutations?
Change in the mRNA codon so a different amino acid is inserted - no detectable change in function.
Non-vital amino acids are substituted resulting in absence of functional phenotypic difference.
What are silent mutations?
Change in the mRNA codon codes for same amino acid to be inserted as in the original coding - no overall effect.
Frameshift mutations
Small scale insertions or deletions can have devastating effect. Insertions add one or more extra nucleotides into the DNA, while deletions remove one or more nucleotides.
If number of nucleotides is not divisible by what ____ it can cause a frameshift mutation?
3
The insertion or deletion can change the what?
Reading frame or grouping of codons, resulting in a completely different translation from the original.
Frameshift mutations normally result in what?
Non-functioning proteins
Why?
because different amino acids are added to the polypeptide after the mutation site
This can lead to the generation of new stop codons, creating a ?
shortened polypeptide
What are 3 common stop codon sequences?
TAA
TGA
TAG
If there is a read-through of normal stop codon what will occur?
a polypeptide chain that’s lengthened
What are 3 common types of DNA variation?
Single Nucleotide Polymorphisms (SNPs)
Variable Number Tandem Repeats (VNTRs)
CopyNumberVariants (CNVs)
What are Single Nucleotide Polymorphisms?
Include synonymous, non-synonymous and nonsense
DNA sequence variation of a single nucleotide
most common type of genetic variation, with 12.8 million SNPs (1:300)
synonymous - don’t result in amino acid change
non-synonymous - result in amino acid change
nonsense - result in a stop codon
What are Variable Number Tandem Repeats?
VNTRs are where a location in the genome has a short nucleotide sequence repeated a number of times.
The micro satellite has how many base pairs?
2-10 base pairs
How many base pairs does the mini satellite have?
Minisatellite: 10-60 base pairs.
What are Variable Number Tandem Repeats useful for?
Useful in genetics and biology research, forensics, and DNA fingerprinting.
What is an example of a Variable Number Tandem Repeats?
the serotonin transporter linked polymorphic region.
What are Copy Number Variations?
They occur how?
larger chunks of DNA missing or duplicated
majority of are inherited; de novo CNVs can occur as a result of mistakes in recombination
Involves deletions or insertions of how many kb/mb ?
1kb- 5mb
What are 2 things CNVs account for?
account for more between-individual base pair variation than any other mutation
account for more differences between humans and other organisms
the influences of pleiotropy and epistasis in disease states
Many human genetic diseases are caused by a single gene mutation that alters the function of an enzyme
True or false?
TRUE
What is Phenylketonuria (PKU)?
- PKU is present in 1 in every 12,000 Caucasian births
- it’s caused by recessive mutations on chromosome 12 in the gene for phenylalanine hydroxylase.
- the body is unable to break down phenylalanine, which builds up in the blood and brain.
- High levels of phenylalanine can damage the brain.
How do you test for PKU in new born babies?
The heel prick test
If positive for PKU what is essential?
A diet of reduced phenylalanine can be used to limit the effects
Toxic levels of phenylalanine, associated with PKU, lead to what?
developmental problems, seizures, hypopigmentation, and a “musty odour” to the baby’s sweat and urine
Explain albanism?
Congenital disorder; complete or partial absence of pigment in the skin, hair and eyes.
Usually caused by a recessive mutation in a gene for tyrosinase, used in the conversion of tyrosine to DOPA, from which melanin derives.
The genes for phenylalanine hydroxylase have pleiotropic effects. What does this mean?
can affect more than one disorder.
What is epistasis?
Epistasis is an important interaction between two or more genes which can affect how they’re expressed.
One gene can mask the phenotypic expression of another.
So no new phenotypes are produced by this type of gene interaction.
PART 2: GWAS
How do researchers identify which genes are associated with a particular phenotype?
GWAS
What is one of the main aims of genetic epidemiology?
One of main aims of genetic epidemiology is to identify the genes or genetic variants whose alleles increase the risk of disease.
the association between genotype and phenotype is tested in a sample of unrelated individuals at millions of what?
Single nucleotide polymorphisms (SNPs)
Why are SNPs used for GWAS?
the most abundant type of polymorphism in the genome.
best chance of finding genetic causes of diseases by testing SNPs
What are 3 problems specific to genetic studies?
confounding caused by both distant and close relatives being in the same sample
effect sizes and statistical power depend on allele frequencies of SNPs
exposure to risk factors can be complicated - sometimes there are several causal alleles in the same region
In GWAS, we test the association between the alleles of a SNP and a trait or disease of interest
True or false?
TRUE
Before we can perform these association tests, we need to code the information of the exposure and the outcome numerically, since we can’t perform statistical tests on letters.
Generally, alleles are coded as what?
0 1
and genotypes are coded as?
0, 1, 2
Why are genotypes coded as 0, 1, 2?
because there are three possible genotypes for each SNP since there are two possible levels on the two chromosomes
So if a SNP can be, for example, T or C,
0 would indicate two copies of the T alleles, 1 would indicate a C allele and a T allele. And 2 would indicate two copies of the C allele. It doesn’t matter exactly how we do the coding.
True or false?
TRUE
genotypes are never coded in a way that reflects the inheritance model. True or false?
FALSE
genotypes are ALWAYS coded in a way that reflects the inheritance model
In 2001, the Human Genome Project provided what?
the complete DNA sequence for one individual
2003 and 2007, three phases of the HapMap Project provided what?
genetic variation data across samples of individuals at millions of SNPs.
facilitated the production of efficient genotyping chips,
Why are genotyping SNPs important?
used to establish what genotype an individual has at over 1 million SNPs across the genome, a process termed genotyping.
Once we have this information across a sample of individuals, we can test to find out which SNPs have genotypes that are associated with the disorder that we are studying.
GWAS search for causal variants associated with phenotypes across the genome
Only prerequisite is what?
that the trait needs to be heritable
The chance that any specific SNP has a causal effect is very low. Thus, we demand a very high level of evidence
Significance threshold is what?
required is P < 5 x 10-8
Usual P-value threshold is 0.05
What is the Key idea underlying the GWAS design?
An allele that predisposes individuals to disease will pass from generation to generation together with SNPs nearby on the genome.
One of these correlated SNPs may have been genotyped and will show an indirect association with the disease, which we can detect in our study.
The statistical power to find genetic variants is dependent on?
Their allele frequencies
GWAS have greater power to find common variants.
True or false?
TRUE
Why does GWAS not work as well with rare alleles?
It is difficult to obtain small P-value results where one allele is rare because its association with the outcome could have occurred by chance
GWAS are now starting to find rarer variants as sample sizes become even larger.
True or false?
TRUE
A Pearson’s chi-squared test statistic for a contingency table with 2 rows and 3 columns has a chi-squared distribution with how many degrees of freedom under the null hypothesis?
2 degrees
If we label the two alleles of a SNP as A and B, where the minor (less common) allele is B, we would code it how?
AA –> 0
AB –> 1
BB –> 2
We are trying to test whether each additional copy of a given allele at a given locus increases risk for a particular phenotype.
True or False?
TRUE
An alternative to analysis based on contingency tables is what?
Regression modelling
What is regression modelling?
- is the most commonly applied statistical technique used for association testing in GWAS
- can be adapted to handle different kinds of phenotype
What are 3 advantages of regression modelling over other testing?
- covariates can be included in the model to correct for confounding and increase statistical power.
- effect sizes can be estimated from the regression coefficients.
- regression models can be used for prediction.
What is logistic regression?
that fit to case-control data
What is linear regression
that look at quantitative phenotypes
Very large x2 statistics indicate a significant association between a SNP and phenotype; however because we are performing hundreds of thousands of tests in GWAS we will expect to find large x2 statistics by chance.
True or false?
TRUE
We can calculate what x2 statistics we would expect by chance over hundreds of thousands of tests, and compare this to what we actually observe. If there were no associations what would we see?
we would see a diagonal line
if there is an over-representation for large x2 statistics above what would be expected by chance, the line would be what?
be elevated above the diagonal line
Larger values on the Y-axis indicate smaller P-values and thus greater evidence for association
True or false?
TRUE
What allows us to visualise data with which regions are associated with disease?
Manhattan Plots
What are 3 techniques for calculating statistical results from GWAS?
Pearson x2 test
Armitage trend test
Regression models
Heritability Estimation
We often talk about heritabilities as a percentage or as a value between what?
0 and 1
So consider a trait in which there are relatively few environmental effects. And most of the trait is being determined by genetic factors.
Would this have a high or low heritability?
High heritability
Alternatively, consider a trait which is strongly under the influence of the environment with relatively weak and few genetic influences
Would this have a high or low heritability?
Low heritability
Why is heritability useful?
Tells us whether we should study a trait
If there is a high heritability. How is it studied?
- Look at DNA to identify specific risk variants and biological pathways causing a disease
- Work towards developing treatment strategies
If there is a low heritability, How is it studied?
Look for environmental risk factors
Focus on policy interventions
The effect of environment can only be measured if individuals vary in their exposure.
True or False?
TRUE
Heritability can change over time.
Heritability may be different at different ages – a trait can have a low heritability in 12 year olds, but a high heritability 25 year olds.
true or false?
TRUE
If we can identify where heritability is low, this can change the way we think about what 3 things?
violent offending
general intelligence
childhood psychiatric disorders
What is Bivariate heritability?
a measure of how much genetic factors predicting traits overlap.
This measure can identify pairs of traits which are genetically very similar and might respond to the same treatment.
What is the percentage of overlap that needs to occur for it to be Bivariate?
33%
What are three methods which use DNA to estimate heritability?
- GCTA
- LDSC
- PRS
Explain GCTA(genome wide complex trait analysis) ?
uses DNA to bypass the need for twins uses data on unrelated individuals
Any pair of individuals in the population has a measurable genetic similarity
What does kinship mean in relation to GCTA?
measurable genetic similarity between individuals in a population.
We can estimate kinship using SNPs.
We sum up the number of SNPs that vary between a pair of individuals, and transform it so that it lies between 0 and 1.
GCTA has 3 advantages over the twin method. What are they?
Only requires genotype and phenotype data from a sample of ~ 2000 individuals
Criticisms about twins being treated differently are not relevant since unrelated individuals are being used here.
By estimating heritability using SNPs, we can focus on genetic risk factors discoverable by GWAS
What does Linkage Disequilibrium demonstrate?
LDSC demonstrates that the maths for GCTA can be rearranged to exploit GWAS results, without the requirement for genotype data.
Is LDSC less or more accurate than GTCA?
is less accurate than GCTA
What is a positive of LDSC?
does not require genotype data at individual level
Explain PRS?
PRS integrates GWAS data and genotype data to test how well genetic variants can predict a trait.
PRS is suited to calculating genetic relationships between pairs of similar disorders.
