Monday, 18 March 2013

Genome-Wide Analysis Using Exon Arrays Demonstratesan Important Role for Expression of Extra-Cellular Matrix, Fibrotic Control and Tissue Remodelling Genes in Dupuytren’s Disease.



A quick guide to the paper:

Genome-Wide Analysis Using Exon Arrays Demonstratesan Important Role for Expression of Extra-Cellular Matrix, Fibrotic Control and Tissue Remodelling Genes in Dupuytren’s Disease.

Authors: Forrester et al


Introduction:

In this paper they are using cells derived from Dupuytren’s patients comparing these cells to similar cells derived from non-Dupuytren’s patients. I have explained this technique before (HERE see the picture about 1/2 way down that has DNA and RNA on it) but I will summarise some of this.

Basically in a cell you have DNA and from DNA something called RNA is created and from an RNA template proteins are made and proteins do most things in the cell. The levels of RNA influence the levels of protein and in the technique they use they basically look at all of the levels of RNA in one cell type (Dupuytren’s) and compare it to another cell type (non-Dupuytren’s). This is then done with as many samples as you can get, in this case 5 Dupuytren’s patients and 6 non-Dupuytren’s patients.

Once you have this analysis you can then group together those that are different and look at what functions they are doing. So for Dupuytren’s (a good example here) it is known that there are high levels of Collagen and therefore you might expect to see an increase in things associated with Collagen production or a decrease in those involved in Collagen break down.

Results:

I am not going to go into too much details with regards to what all these things do, there will hopefully be enough detail so that you can either go to the paper and understand it better and or go to Wikipedia and try to get a better understanding but it all likelihood it will not mean that much to anyone as this is a very specialised field which I find hard to understand, so unless you have a degree in Molecular Biology it will be very very hard.

In the Dupuytren’s cells they found that there were:
·         307 genes up-regulated (with high RNA levels)
·         1288 genes down regulated (with low RNA levels)

Many of the genes in this above set were associated with the Extra Cellular Matrix (ECM – area where Collagen is and problem area for Dupuytren’s patients) but there were also changed in many other pathways such as growth factors and tissue remodelling e.g. collagenases (those that break down collagen) and matrix metallopeptidase proteins or MMPs (also previously linked to DD and break down collagen).

Down regulation of MMPs (which break down Collagen):

I will start as the paper did with those that were most different between the 2 groups. The MMPs, these showed a 56 fold reduction in expression (RNA) levels, as you can imagine if you have a balance between the production and breakdown of a substance and you remove a major part of that breakdown you will get more and more of the product. I guess a way of imagining this might be that you have a ½ full bucket (the normal level, with water representing RNA) and into this bucket you have a steady supply of water at 1L per minute but you have holes (the proteins that break down the Collagen) in the bottom and you are losing 1L / minute in a normal bucket but in a Dupuytren’s bucket you have 50 times less of those holes in the bottom so you are losing only 20ml / minute, as you can imagine this means the bucket will gain 980ml of water / minute and will quickly overflow. The same principle applies to the Dupuytren’s cells; the collagen keeps being made but is not being broken down.
     
Collagen:

Conversely to MMPs the levels of Collagen expression were shown to be greatly up regulated, so not only do you have less water coming out but you also have more water going into the bucket.

Cell adhesion genes:

So there are also genes that are coding for proteins that help other proteins to stick together and ones that stop them sticking together, as you would expect for DD, where the cells clump together to form lumps, you get an increase in those that help others to stick together and a decrease in those helping everything fall apart. The cell is very much about maintaining a balance and the common theme for DD cells is that they are no doing that correctly.

Fibroblast growth factors:

The cells that produce the collagen are called fibroblasts and like everything else these are regulated by a finely tuned process. In the body there are many different growth factors, some of which are fairly famous due to their use by sports cheats and there link to dwarfism etc (growth hormone) or a link to ageing (IGF-1). The main hormone that controls fibroblast growth is (not surprisingly) Fibroblast growth factor (of which there are many), it was noted that in DD cells this is expressed at levels 12 times that seen in normal cells.

We could take this back to the bucket, if you think of each bucket as a cell producing collagen, but that the number of buckets you have is determined by how much money you have, DD patients have a lot of income (we wish) and so we have lots of money (i.e. growth factor) and therefore we have lots of buckets and we are replacing them quicker than they are getting broken and they are all are already making too much water so DD hands (and potentially LD feet) are being flooded with cells which are making too much collagen and so they produce lumps and cords.   

Discussion:

One of the unique, or at least rarer points of this paper, is that they are using normal cells from different patients from a different part of the body, most studies of this kind either use non-diseased tissue from the patient or from a carpel tunnel patients. This is good and bad, you could argue that getting tissue as similar as possible to the disease tissue is important (so non-disease from patient is best) but also that this may be too varied and so there may be some things that are missed, so the approach of this paper works best. To be honest the discussion is very technical and scientific and if you want to read it I recommend looking at the paper as it will take me too long to reproduce here. Basically they show that there is an up regulation in collagen and collagen related factors are down / up regulated in DD patients and this is not really surprising as you see high levels of Collagen in DD patients.

To quote their conclusion:

“In conclusion, we have comprehensively characterized the transcription profile differences between DD and normal primary fibroblasts. Our data indicate that in DD there is an excess of collagen and other ECM that is not controlled due to a reduction in matrix metalloproteinases and other matrix remodelling proteins. A reduction in the fibrotic control protein, follistatin, may also contribute to DD. In addition, the fibroblasts lack expression of genes involved in cell movement and cytoskeletal organisation and an increase in genes involved in cell adhesion. These indicate a lack of organisation of both extra- and intracellular matrix as well as a lack of cellular movement in DD. Alternative transcripts have also been identified which are expressed at different levels in the DD patients compared to the controls and may reflect cell stress such as hypoxia. These conclusions will be the basis for future experimentation. Many of the identified genes are potential candidates for the treatment of DD. There was a close correlation between expression levels in some genes from our study and data from previous studies using DD tissue samples providing reason to pursue investigations into potential therapeutic development strategies using in vitro studies on DD fibroblasts. It is likely some of these candidate genes for treating DD will also be effective for fibrotic diseases in general, including injury-related and radiotherapy-induced fibrosis.”  

Also posted on the BDS blog: http://dupuytrenssociety.blogspot.co.uk/