Genome variation

Question 1

What is karyotyping?

Answer 1

the process of sorting chromosomes into their matched pairs

an important technique for investigating chromosomal disorders

Question 2

What are Macro-level differences in genome generally associated with?

Answer 2

Generally associated with disease:

• aneuploidy
• translocations

Question 3

What are Micro or molecular differences in genome sometimes associated with?

Answer 3

Sometimes associated with disease:

• point mutation causing sickle cell anaemia
• 3bp deletion in CFTR

Question 4

Which regions of a genome affect trait?

Answer 4

Exome region (about 2% of the genome)

the small proportion of variation accounts for all the genetic difference that then leads to phenotypic differences (the results in phenotype that we get is a combination of DNA and the environment, not solely DNA variation)

Question 5

What is a polymorphism?

Answer 5

position on the genome that varies between individuals (a variant)

Question 6

What is a reference sequence?

Answer 6

consensus sequence that tells us what we expect to see in the genome

this is used to determine whether there are any genetic variants (polymorphisms)

Question 7

What are Single Nucleotide Variants (SNVs)/Polymorphism (SNP)?

Answer 7

Substitution of a single nucleotide at a specific position in the genome.

High in frequency and occurs 1 in 300 nucleotides in reference genome

Bi-allelic

In every individual, occurs once in every 1000 bases (1kb)

Question 8

How are SNVs/SNPs generated?

Answer 8

During mismatch repair during DNA replication:

1) Mismatch during DNA replication
2) DNA mismatch repair system should excise out the base that doesn’t belong there
3) Instead, it incorrectly excises out the wrong base, resulting in genetic variation

Question 9

Where are the majority of SNPs?

Answer 9

Not in the exome

Question 10

Why are the majority of SNPs not in the exome?

Answer 10

because exome has strong selective pressure to not incoporate mutations which will lead to detriment in individuals

Question 11

Describe the function of the selective pressure in the exome.

Answer 11

high selective pressure in the exome keeps the variation relatively low in comparison to the non-coding regions of the genome

HOWEVER, there still is variation in the exome causing for the variations amongst individuals (e.g. hair colour, height, intelligence, etc)

Question 12

Where are SNVs found?

Answer 12

Gene
>no amino acid change (synonymous)
>amino acid change (non-synonymous/missense)
>stop codon (nonsense)
>splice site
>UTR (gene expression)

Promoter
> affects protein expression

Non-Coding Region
>if in non-coding region, less likely to have a detrimental effect

Question 13

What is the only way SNVs disappear?

Answer 13

If they have a deleterious effect or population annihilation.

Without a deleterious effect or population annihilation, SNVs do not disappear

Question 14

What is sickle cell anaemia caused by?

Answer 14

Single nucleotide variant (point mutation: non-synonymous/missense) changing the base sequence and therefore the amino acid sequence.

GAG → GTG
Glutamic Acid → Valine

Question 15

What is the Link between sickle cell anaemia and malaria?

Answer 15

Individuals who are heterozygous for SCA have some protective advantage against malaria. As a result, the frequencies of sickle cell carriers are high in malaria-endemic areas

SCA point mutation is therefore very rare in white European population (0.02%), but relatively common in black African population (4.5%)

Question 16

When is an SNV described as a polymorphism or a mutation?

Answer 16

SNV is described as a:

• polymorphism if minor allele frequency is >1%
• mutation if minor allele frequency is <1%

HOWEVER, this doesn’t always hold true:

• rare polymorphism: MAF 1-5%
• common polymorphism: MAF>5%

Question 17

What do SNVs arise from?

Answer 17

All SNVs arise from a mutation and so are rare to begin with

Question 18

How do SNVs spread throughout the population?

Answer 18

through reproduction and migration unless there is negative selection pressure against these variants

Question 19

What is positive pressure selection?

Answer 19

genotypes that result in a beneficial trait that increases fitness are chosen FOR

Question 20

What is negative pressure selection?

Answer 20

genotypes that result in a disadvantageous trait that reduces fitness are chosen AGAINST

Question 21

What are SNVs called when proven to be pathogenic?

Answer 21

Point mutations

Question 22

Describe how evolution is tied with SNVs.

Answer 22

1) New allele arises from mutation, causing a variant
2) Gene flow through reproduction and migration leading to introduction of that variant into another population
3) Genetic drift as there is a random change in variant allele frequency between generation
4) Selection: non-random change in variant allele frequency between generations because presence of one allele/genotype is pathogenic (negative selection) or beneficial (positive selection)

Question 23

What are Microsatellites (short tandem repeats)?

Answer 23

repetitive DNA units typically 2-5 nucleotides in length, which directly affect the physical length of the genome

unlike SNVs which are bi-allelic, micro-satellites are tri-allelic

Question 24

Why do microsatellites arise?

Answer 24

due to a problem in DNA replication (Polymerase Slippage):

1) DNA Polymerase pauses to proofread, and elongating strand becomes disengaged from template
2) Elongating strand needs to re-anneal to template so polymerase can continue adding specific nucleotides, but because template strand consists of repetitive sequences, elongating strand re-anneals to incorrect base pairs because they are all identical
3) This causes a bubble of unpaired bases
4) DNA Repair Mechanism adds two extra bases into the parental strand and elongation therefore continues

Question 25

Where does polymerase slippage occur?

Answer 25

only in sections of DNA with repeated patterns of bases and cannot occur in DNA without repeating patterns of bases

Question 26

Where are microsatellites found?

Answer 26

May be part of 98% of the genome not coding for a protein:

• Intronic or UTR: may affect gene expression
• Intergenic

May also be exonic
>causes extra amino acids in protein and results in expansion disorders such as Huntington’s= trinucleotide repeat expansion disorder

Question 27

What characteristic do STRs have that allows them to be used for identification of an individual?

Answer 27

Each STR is highly polymorphic

Question 28

How is PCR used to amplify STRs?

Answer 28

unique primers flanking each STR allow specific PCR amplification of the two STR alleles at a given locus

Question 29

How do STRs identify an individual in the UK database?

Answer 29

A panel of 10 STRs that are used in the UK are amplified and are sorted by size (gel electrophoresis) and the repeat number is thus determined.

This gives a pattern of 20 numbers along with a gender identifier, and this gives a uniqueness (frequency) of 1 in 1 billion.

Question 30

What is a copy number variant (CNV)?

Answer 30

a polymorphism in which the number of repeats of a DNA sequence at a location varies from person to person

Question 31

What is the simplest type of CNV?

Answer 31

presence or absence of a gene

-individual’s genome could therefore contain 2 (one on each homologous chromosome), 1 or 0 copies

Question 32

What is a CNV caused by?

Answer 32

CNV is caused by non-allelic homologous recombination in meiosis
>allelic recombination is good because it increases genetic variation, however non-allelic recombination results in duplication/deletion and copy number change, impacting future generation

Question 33

What is Non allelic homologous recombination?

Answer 33

a form of homologous recombination that occurs between two lengths of DNA that have high sequence similarity, but are not alleles

Question 34

When are CNVs pathogenic?

Answer 34

CNVs are pathogenic in microdeletion disorders e.g. diGeorge syndrome

Question 35

What are the effects of most common variants?

Answer 35

Most common variants don’t cause Mendelian monogenic disorders and the majority are probably neutral (particularly intergenic variants)

BUT, they may well impact upon non-Mendelian disorders and undoubtedly contribute to general individual variation (e.g. personality, sporting ability, looks, etc)

Variants therefore can be:

• beneficial
• pathogenic
• neutral

Question 36

Give clinical uses of variants.

Answer 36

Used as markers to help find disease-causing genes and mutations:

• Autozygosity Mapping and Linkage Studies (microsatellites, SNPs)
• Association Analysis (SNPs, CNVs)