Raju Kucherlapati, M.D.
Raju Kucherlapati, M.D., Professor and Chairman of Molecular Genetics, Albert Einstein College of Medicine, New York: "Efforts to clone the Noonan Syndrome gene"
These are actually international efforts to try to clone the gene for Noonan Syndrome.
Noonan Syndrome is inherited as an autosomal dominant disorder (autosomes are non-sex chromosomes). A dominant disorder indicates that if either one of the two copies of each gene that we carry is mutated or modified, then such individuals will display signs of the disorder. Many of these will result from single changes in one nucleotide in the genome. So why is it difficult to try to isolate the gene?
All of us carry genetic information that dictates what we are, and this information is composed of three billion base pairs of DNA. And so what we are looking for in patients with NS is to see if one of those three billion base pairs of DNA might be different, and then to try to identify which one is different and causes the disorder.
This is complicated by the fact that we are all different, because we all carry a unique genetic content which provides us with our individuality. So when we are looking for the difference which causes NS, we are looking for that one base change in a sea of differences that we find in the national population.
Fortunately there are methods which help us to take shortcuts to try to find the gene, and these shortcuts are known as positional cloning. Even if you don't know exactly what the gene is doing, if you know the position of the gene within the genome then it is possible to be able to identify it.
What we are trying to do has also been helped greatly by the human genome effort that was initiated around 7 years ago by the US government and other governments internationally. The goal of this was to be able to try to identify all of the genes that are encoded by the human genome and to actually have the sequence of the DNA that is present in the human body. It is anticipated that when the complete human genome sequence is available we will really be able to understand all of the genes that are present in the human body, and that would facilitate the identification of genes involved in NS as well as many other disorders.
The first step in this process is to try to identify the region of the genome where the gene for Noonan's is located. The approach used to try to identify the correct region is known as linkage analysis, and this process starts with the identification of families in which there are several patients with NS, and trying to establish whether the syndrome is associated with particular types of markers.
There are a number of different types of genetic markers which allow us to identify which portions of the genome are associated with NS. At least one major gene associated with NS is located on chromosome 12.
An international consortium (in the Netherlands, London, and New York) is trying to identify the gene. This is very important since if the different research teams were working in competition to identify genes, important information may not shared, so several researchers working on the program decided that pooling resources was important.
The Netherlands group is aiming to identify large families in which NS present. They are then using linkage analysis to locate the affected area gene(s).
The London team are being presented with candidate genes and are looking for changes in genes which might be responsible
The New York team are actually trying to identify all of the genes on 12 which may be candidates.
First step of positional cloning is to map to find out where the gene may be located. It is know that the gene is on chromosome 12, but we need to identify which of many genes present in that region is responsible. Then there are technologies available to identify what the function of the gene is, and why changes in that gene should cause NS. The hope is that if we understand the function of an affected gene, it would facilitate in either management or therapy of the condition.
So the strategy to clone the gene is to identify large affected families and to ascertain by linkage analysis the position on the genome where gene located. There is therefore a continuous need to identify NS families.
Once located, it is important to try to actually isolate or clone a fragment of DNA that comes from that region and to be able to identify all the genes present and to analyse them by mutational analysis - to look for changes in genes.
The first process is the construction of a map of the region, and the goal is to try to identify the DNA that encodes this region of chromosome 12 from one end to the other. This is done by taking fragments of DNA isolated from the chromosome and sequencing them; overlapping regions of fragments allow sequencing of the whole chromosome, since markers on the map can be used for orientation just like street names.
Mapping efforts are currently underway, and so far linkage analysis been able to localise the region from the 3 billion base pairs to 5 million base pairs. However, it is still necessary to reduce this further; narrowing it down to 1 million base pairs would facilitate things a lot. The efforts to clone and isolate DNA in this region is nearly complete.
Once the regions of interest have been identified, the DNA is sequenced to identify all the genes present.
It was mentioned that the region of interest focussed on 5 million base pairs; of these, fragments of around 2.5 million base pairs have been examined, of which around 70% have been sequenced.
Around 1 million base pairs can contain about 30 genes, so the 5 million base pairs being studied will contain around 150 genes, and of the total number discovered so far, about 105 have been identified.
Once identified, we can look at genes and see what they are, and from knowledge of NS and associated phenotypes we can look at a list of genes and decide if there are any important ones that we want to look at. We then need to look for consistent differences in these genes between NS and normal individuals; this is very time consuming.
One gene of interest is TBX5, a T-box gene involved in development, which 3 years ago was shown to be mutated in Holt-Oram syndrome, which causes abnormalities of the heart and limbs. We are therefore looking to see if other members of this family of genes may be located in the area of chromosome 12 which we are studying.
Another gene, MYL1 (myosin light chain), is expressed specifically in muscle and may be another that needs to be looked at, although none of the NS patients have been found to have mutations in that gene.
There are a number of these types of genes, but as yet none stand out as being likely to be involved in Noonan Syndrome. So although we want to be able to go ahead and look at all these genes, we need to continue identifying other genes in this interval which may stand out as possible candidates. We have identified two of these genes (MYL1 and ribosomal protein L6) in this critical region, but they are normal in NS and can be excluded.
We are really looking at a rather large region of chromosome, and although we are committed to examining the whole of this it would be nice to narrow the size of the region. For this reason we are still trying to collect additional families to use in the studies.
We are continuing to evaluate candidates as new genes are identified, and to establish whether any may be involved in the tissues or organ systems involved in Noonan Syndrome, as this will help in evaluating candidate genes. We need to look for changes in these genes in Noonan's patients and to understand which of these genes are involved.
Finally, a more complex scenario is that it is not only the gene on chromosome 12 that is responsible, but that the product of that gene may interact with other genes that are encoded by other portions of the genome; this might actually explain the variability seen in Noonan Syndrome. However, this is a much more complex situation to try to figure out.
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