Rare Diseases RNA Phenotyping Project

DNA (deoxyribonucleic acid) is your genetic code; it is present in almost every cell in your body and contains the instructions to be able to make every part of you.​ However, although it is present in almost all cells, most of it does not need to be ‘read’. Your brain cells, for example, will just need to ‘read’ and express the parts of the DNA that are important for the brain cells to function. It will not ‘read’ or use (or express) parts of the DNA that are unique to, for example, skin cells.​ 

 RNA (ribonucleic acid) is a genetic copy or ‘read out’ of the parts of the DNA that are actively being used (expressed) by the cell. These are then generally translated into protein and result in the cells’ functionality.​  

 Proteins are translated from RNA; they are biological molecules that are essential for life and are made up of subunits called amino acids. These amino acids are coded in the DNA and therefore the RNA as well. So, if there are variations in the DNA and RNA codes then this impacts the protein’s structure and potentially its function. These differences may play a role in diseases and give us an insight into disease mechanisms. 

Genetics: the study of single genes and how they are inherited.  Genetic tests are able to identify a specific gene or variation in a family.  

Genomics:  the study of all the genes (also called the genome) at once, as well as the interaction of those genes with each other and any environmental influence on these genes.  A genomic test can compare more than one gene at a time and in some cases all the DNA (Whole Genome Sequencing).  

Whole Genome Sequencing gives a ‘read out’ of all your DNA – your genome. Your DNA sequence is unique to you, however, some of it is shared with your relatives. By comparing your genome and understanding how it is similar or different from others, it is possible to learn more about why diseases may occur and how they may affect you. 

RNA is a molecule similar to DNA, but unlike DNA, RNA is single-stranded and is short-lived within the cell. RNA plays an active role in cells, regulating when and how much genes are expressed which could have a role in disease mechanisms.  

Around 1,000 people's RNA with specific rare diseases will be studied using scientific methods that will allow for the discovery of new genetic disease associations and mechanisms. Involvement in this study might not benefit the participants directly, however it may lead to an overall improvement in understanding of the rare disease. 

Neutrophils  

Neutrophils are very abundant white blood cells, with ~100 billion produced in our bone marrow every day. They constantly patrol the body for microorganisms and are the first white blood cells to arrive at the site of infection. They then respond by trapping and killing the invader such as during bacterial infections. They can do this by ingesting the microorganism and releasing molecules that kill it. Therefore, they are an essential part of the immune system.  

Monocytes  

Monocytes are very flexible white blood cells which respond to signals within the human body and ultimately give rise to cells that help to resolve cell damage. Monocytes also recruit other cell types to affected sites, mounting an immune response. They have been shown to play a role in a number of conditions including cancer, neurodegenerative diseases and obesity. 

T Helper cells  

There are several types of T Helper cells that carry out a variety of immune functions. These include helping other immune cells to respond to inflammation and infection.  Other types of white blood cells, called B cells, are triggered to make antibodies, some of which can remember infections so that they can respond rapidly to future re-infections and prevent the infection from spreading. 

Platelets  

These cells are tiny pieces of cells called a megakaryocyte that originate in the bone marrow. Platelets are replaced every 5-7 days and are key to help blood clot, stop bleeding and heal wounds. They do this by 'sticking' to the area that is damaged and creating a platelet plug or clot.  

Your 50ml blood sample arrives in the laboratory, it is important that we start processing the sample as soon as possible as it continues to change outside of the body. 

When the blood arrives in the laboratory, we keep a little to one side which we use to extract DNA from later. Then with the rest of the blood we need to separate out the plasma. Plasma is a light-yellow liquid which contains the platelet cells.  As it is lighter than the rest of the blood, we spin the whole blood at high speeds of 200g-force. This means that the lighter plasma rises to the top while the rest of the blood sinks to the bottom of the tube. We can then remove the plasma from the rest of the blood. 

Now we have the plasma separated we spin this again but this time at even higher speeds of 1500g-force. This means that the platelets sink to the bottom of the tube and we can separate out the platelets from the rest of the plasma. We then freeze down both the platelets and the plasma. 

With the rest of blood that remains from the first spin we now separate and remove the red blood cells as we are not interested in these cells for this project. We do this by adding a thick gel to the bottom of a tube, we then carefully layer the remaining blood on top of this thick gel. We spin this at 1200g-force, this results in the red blood cells sinking and getting ‘trapped’ in the thick gel. We can then pour off the top layer that contains white blood cells This includes the neutrophil, monocyte and T helper cells that we are interested in. 

As these cells are currently all mixed together, we need to separate them out so that we can look at them individually. To be able to do this we use a cutting-edge technique that uses tiny magnetic beads. These beads have special tags on them that mean that they attach to a specific cell type. Therefore, we add beads that attach to one of the cell types and then place the tube on a magnet. The beads will move to the side of the tube towards the magnet with the cells of interest attached to them. We then remove the liquid from the tube and the beads stay within the tube ‘attached’ to the magnet. We then add some fresh liquid to the tube with the beads and cells of interest in. These can now be frozen and stored at -80°C. 

We repeat the process of adding magnetic beads for the other cell types of interest until we have all the cells frozen and stored. This is the end of the first initial day of processing. 

On the second day of laboratory processing, we extract the RNA from the cells. Firstly, we need to break open the cells. We do this by adding a chemical and then mixing the cells vigorously by shaking them. We then spin the cells at high speeds of 1200g-force, which separates out the liquid into 3 distinct layers. We remove the top later that contains the RNA. We now need to clean up the RNA and remove any other molecules that may be contaminating it. So, we add alcohol to the tube and the RNA solidifies at the bottom so that we can then pour off the liquid.  Now we have pure RNA and we add some fresh liquid to the tubes to rehydrate it. 

This RNA is now measured for purity and yield and can then be prepared for sequencing. DNA extraction is a similar process to extracting RNA and is performed on the blood that was set aside right at the start. 

Once the RNA and DNA has been sequenced, we get lots of files with all the data. This data is then analysed by bioinformaticians (computer scientists that specialise in looking at biological data).

When we have data from lots of individuals with the same rare disease we can look to see if there is something similar that is happening in their data and if this is different to healthy control participants. If there is, we can look to see if this might help explain the rare disease and what impact this knowledge may have.