Genomic imprinting and dynamic mutation

Question 1

What is genomic imprinting?

Answer 1

Where in the somatic cells an individual only expresses one of their inherited alleles (either the maternal or the paternal one). One of the copies of a gene is turned off/silenced.

Normally we express both of these alleles.

AN IMPRINTED ALLELE = A SILENCED ALLELE

Question 2

Why is genomic imprinting refered to as an epigenetic phenonanon?

Answer 2

Genomic imprinting is a heritable phenotype resulting from changes to the chromosomes without any actual DNA change/mutation. (IT OPERATES IN THE PROMOTER REGION)

Eg this may be from a methyl group being added to cytosine in the promoter region of a gene which then alters the expression

Question 3

Approx how many genes in the mammalian genome show imprinting?

Answer 3

0.1% (very few)

Question 4

What must occur in order for syndromes to happen as a result of abnormalities in an imprinted region of a chromosome?

Answer 4

There must be a mutation (a deletion) of one of the parental copies of a gene and the other copy must be imprinted on (and therefore silenced).

Question 5

What is the difference between prada willi syndrome and angelmans syndrome?

Answer 5

Both are genetic disorders resulting from loss of expression to a region of chromosome 15.

Prada willi syndrome is a result of loss of deletion of the paternal copy of the gene in combination with the maternal copy being imprinted on (and therefore silenced).

Anglemans syndrome (happy puppet syndrome) is a result of deletion of the maternal copy of the gene in combination with the paternal copy being imprinted on (and therefore silenced).

Question 6

What are some of the characteristics which could help differentiate between prada willi and angelmans syndrome?

Answer 6

Angelmans syndrome =
developmental delay and ataxia (inability to crawl/walk/balance/talk) BUT they are very happy children and tend to laugh/smile a lot

Prada willi syndrome =
failure to thrive, initial feeding difficulties and loss of appetite followed by overeating and rapid weight gain/potentially obesity after 1 year. Often very small hands and feet and very short stature.

Question 7

What is UPD? and how can this potentially cause one of the above syndromes?

Answer 7

Uni-parental disomy =
Uniparental disomy refers to the situation in which 2 copies of a chromosome or two parts of a chromosome come from the same parent, instead of 1 copy coming from the mother, and 1 copy coming from the father. Angelman syndrome (AS) and Prader-Willi syndrome (PWS) are examples of disorders that can be caused by uniparental disomy.

Question 8

How are genes normally expressed in individuals and what is the difference with imprinted genes?

Answer 8

In diploid organisms (like humans), the somatic cells possess two copies of the genome, one inherited from the father and one from the mother. Each autosomal gene is therefore represented by two copies, or alleles, with one copy inherited from each parent at fertilization. For the vast majority of autosomal genes, expression occurs from both alleles simultaneously. In mammals, however, a small proportion (

Question 9

When does imprinting occur in the maternal and paternal gametes?

Answer 9

Methylation changes are “imprinted” on the germline cells (sperm or egg cells) which means they are inherited and will be present in all somatic cells of the offspring.

In mothers this occurs during oocyte formation.
In fathers this occurs prior to meiosis in the primary spermatocyte.

Question 10

Where does the methylation occur in the chromosome?

Answer 10

In the promoter region on cytosine residues within CpG islands. CpG islands are big regions of base pairs in the promoter region that contain lots of G and C residues.

Question 11

What is the result of CpG methylation of gene promoters?

Answer 11

Transcriptional silencing - that copy of the gene will not be expressed.

Question 12

Can methylation be reversed?

Answer 12

Yes it can be reversed in the early embryo.

This is achieved by inhibiting the methyltransferase enzyme which allows the methylation to be transmitted through cell divisions.

Question 13

Summary of genomic imprinting to just read.

Answer 13

We have evolved regions in the promoter regions of many of our genes called CpG islands which allow methyl groups to be applied to cytosine residues. This regulates how many genes are expressed.

This is important in the inheritance of imprints. Imprints occur when we get methylation of one of the copies of a gene (either the maternal or paternal) which results in silencing of that copy and so only one allele is expressed. This results in syndromes such as anglemans syndrome or prada willi syndrome depending on which parent chromosome the imprinting occurs on and it also must happen in conjunction with a mutation to the other allele result in complete loss of that gene or set of genes.

Question 14

What are the two steps involved in the formation of cancer as a result of imprinting?

Answer 14

1. Inherited predisposition =
   Methylation (imprinting) of a promoter region of a tumor suppressive gene from one parent may/will silence it
2. Somatic methylation
   One you have already inherited a silence tumour suppressor gene from one parent then that gene is much more vulnerable to be turned off by a somatic cell mutation that affects the other copy

Question 15

What is an example of a syndrome caused by DNA methylation that is linked to cancer?

Answer 15

Beckwith - wiedemann syndrome

An overgrowth condition that causes increased tumor predisposition (wilms tumour)

It is largely due to excess of paternally expressed growth factor and lack of maternally expressed tumor suppressor.

Question 16

What is dynamic mutation?

Answer 16

Dynamic mutation is where a gene is “instable” and there is progressive exspansion of repeat sequences in a gene - this is often “triplet repeats” eg in Huntingtons disease there is repetition of CAG in chormosome 4.

Question 17

What is the relationship between the number of repeats and the severity of the disease?

Answer 17

There is a general trend of the more repeats you have the more severe your symptoms will be.

Generally the larger the expansion the more likely they are to cause disease or increase the severity of disease.

Question 18

What is the phenomenon of anticipation in terms of dynamic mutation?

Answer 18

Anticipation refers to the fact that in each successive generation that inherits this dynamic mutation there is an earlier age of onset and more and more triplets are inherited so there is more severe symptoms.

Question 19

How does PCR work? (polymerase chain reaction)

Answer 19

Sample Preparation

Before initiating PCR, DNA must be isolated from a sample. DNA extraction is a multi-step process that may be done manually or with an instrument like the COBAS® AmpliPrep Instrument, the first instrument that prepared samples automatically without human intervention.
Following sample preparation, the three-step PCR process is initiated.

1. Separating the Target DNA - Denaturation

During the first step of PCR, called denaturation, the tube containing the sample DNA is heated to more than 90 degrees Celsius (194 degrees Fahrenheit), which separates the double-stranded DNA into two separate strands. The high temperature breaks the relatively weak bonds between the nucleotides that form the DNA code.

2. Binding Primers to the DNA Sequence - Annealing

PCR does not copy the all of the DNA in the sample. It copies only a very specific sequence of genetic code, targeted by the PCR primers. For example, Chlamydia has a unique pattern of nucleotides specific to the bacteria. The PCR will copy only the specific DNA sequences that are present in Chlamydia and absent from other bacterial species. To do this, PCR uses primers, man-made oligonucleotides (short pieces of synthetic DNA) that bind, or anneal, only to sequences on either side of the target DNA region.

Two primers are used in step two - one for each of the newly separated single DNA strands. The primers bind to the beginning of the sequence that will be copied, marking off the sequence for step three. During step two, the tube is cooled and primer binding occurs between 40 and 60 degrees Celsius (104 – 140 degrees Fahrenheit).

Step two yields two separate strands of DNA, with sequences marked off by primers. The two strands are ready to be copied.

3. Making a Copy - Extension

In the third phase of the reaction, called extension, the temperature is increased to approximately 72 degrees Celsius (161.5 degrees Fahrenheit). Beginning at the regions marked by the primers, nucleotides in the solution are added to the annealed primers by the DNA polymerase to create a new strand of DNA complementary to each of the single template strands.

After completing the extension, two identical copies of the original DNA have been made.

Cycles of AmplificationAfter making two copies of the DNA through PCR, the cycle begins again, this time using the new duplicated DNA. Each duplicate creates two new copies and after approximately 30 or 40 PCR cycles, more than one billion copies of the original DNA segment have been made. Because the PCR process is automated, it can be completed in just a few hours.

In a healthcare setting, PCR makes enough copies of target DNA from the clinical sample to allow analysis; the results of these diagnostic and monitoring tests provide clinicians and other healthcare providers with information to guide treatment.