By Serge B. Melanšon, M.D.
Andrea Richter, Ph.D.

The research team on Hereditary Ataxias of the medical genetics laboratory at Saint-Justine's Hospital just accomplished a canadian premiere in pre-natal diagnosis: the direct molecular analysis of amniotic cells for a pre-natal diagnosis of Freidreich's Ataxia.

Up to now, the pre-natal diagnosis for this serious disease involved multiple steps, including the analysis of the patients' and their family's DNA with molecular probes that detect restriction fragments length polymorphism (RFLP).

This method required many weeks of intensive laboratory work. Moreover, the results were not always applicable to pre-natal diagnosis. Thus, it is not surprising to observe that only one result of pre-natal diagnosis based on this method has been published since the localization of the gene for this disease in 1988.

Thanks to the research done in the last year, the team from Sainte-Justine's Hospital benefits from new molecular probes extremely informative, developed in the laboratories of Drs Susan Chamberlain in London and Jean-Louis Mandel in Strasbourg.

Those new markers, located very close to the FA gene, enables us to analyze directly the DNA of people at risk from a very small quantity of cells, such as found in the amniotic liquid.

This is how, in less than two weeks, Quebec researchers were able to complete the molecular study of a family at risk for FA, and to do a pre-natal diagnosis without having to culture amniotic cells, as it is the case for most genetic pre-natal diagnosis.

Instead of waiting 3 or 4 weeks, as it was the case before, the results from the pre-natal diagnosis was transmitted to the couple within 48 hours following the amniocenteses.

This technologic development is a valuable gain for the continuation of research supported by CAFA for identifying the genetic default that causes this disease.

Translated by Fanny Chagnon

Friedreich Ataxia Research Update

Massimo Pandolfo, MD
Montreal, Quebec, Canada

Dr. Pandolfo received his M.D. at the University of Milan, Italy in 1980 and his post doctorate in molecular genetics from the University of California, Irvine.
From 1988 to 1993, he worked in the Division of Biochemistry and Genetics of the Nervous System at the National Neurological Institute in Milan, Italy.

From 1994 to 1996, he served as Assistant Professor of Neurology at Baylor College of Medicine in Houston, Texas. Since 1996, he has served as an Adjunct Professor at McGill University, Department of Neurology and Neurosurgery, in Montreal Canada. He also serves as Research Associate Professor in the Department of Medicine. Dr. Pandolfo, working in collaboration with other researchers, discovered the Friedreich Ataxia gene in 1996.

I am going to give an overview of Friedreich ataxia over the past year. I also want to mention the important discovery of a new recessive ataxia gene, ARSACS (Autosomal Recessive Spastic Ataxia of Charlevoix Sanguenay) .
Credit for this discovery should be given to Andrea Richter from Montreal. Dr. Richter has dedicated many years to mapping and identifying this gene.
ARSACS is a very rare disease found in a remote northeastern part of the North American Continent (Charlevoix Sanguenay region of Quebec).
This is a recessive, spastic ataxia that is probably found elsewhere in the world. There have been descriptions of cases that resemble it clinically and the gene maps to the same chromosome region, so we assume it is the same in families from Tunisia.
It is likely this disease will be found with some frequency in other families of European and North African origin. The gene is a novel gene that doesn't really resemble anything with a known function.
We know that it is highly expressed in the cerebellum, and that the cerebellum becomes severely atrophic.

Finding genes for genetic diseases is a little like finding the black box after an airplane crash. We find the "black box" that is essential to understand what went wrong, but it still needs to be decoded to understand what exactly went wrong and what measures we can take so this disaster does not happen again. When we find genes we find the primary abnormality in a genetic disease.
We still have to go through all of the steps from the gene mutation to the development of the disease in order to understand what measures we can take to stop and prevent the disease from developing in younger persons who are at risk.

How are we doing in understanding the data in the "black box" for FA? There has been substantial progress in the past year, at all levels, from understanding the mutation at the DNA level that caused the disease to having some clues about the function of the protein that is encoded by the gene.
We are defining its function; we are starting to have some idea of the cellular processes that go wrong. And, some treatments are being proposed in order to correct this defect. There are many laboratories that are actively working on generating an animal model for this disease. I think we are close to getting it.

FA is caused by the expansion of a GAA triplet repeat in the Frataxin gene. This disease is probably restricted to people of European, North African, and Middle Eastern origin. The disease is essentially absent from east Asia, not found in China, Japan, SE Asia, or Africa south of the Sahara, and is not found in Native Americans. It is what we call a Indo-European disease. We also have an idea why.
We find the longer repeats (that can become at risk to expand) only in people essentially from North Africa and Europe. Basically, for some reason this mutation has only been introduced to this population, probably when people were starting to migrate out of Africa. This is not only a curiosity, but is also important when making diagnosis for people of different backgrounds.

Through a collaboration between us and Houston, Texas, A and M University, Institute of Biosciences and Technology, and the University of North Carolina Chapel Hill, it was discovered that the repeat that causes FA forms a tangle in the DNA molecule. It is called "sticky" DNA because it makes two DNA molecules stick to each other in the region that the GAA repeat is contained.
We interpret this to mean that this tangle of DNA strand is what causes the problem in patients because it prevents the gene from being properly expressed. For a gene to be expressed, a copy of the DNA molecule has to be made into a different acid, RNA, then the RNA copy of DNA is used to direct the synthesis of the protein that is encoded by the gene.
To copy a segment of DNA into RNA, a molecule called RNA polymerase slides along the DNA molecule and synthesizes a copy of it. If RNA polymerase reaches a tangle, it will probably stop and have a hard time getting through it. This is probably what is causing the disease.

This is important for all molecular biologists and patients for several reasons. First because it is progress in the understanding of the molecular basis of the disease. Second, with this discovery came the discovery that just changing a few bases in the repeat sequence can completely destabilize the structure and allow gene expression to proceed normally.
This is a possibility for the future; to intervene in the disease by trying to introduce changes in this sequence and destabilize it. This is not going to happen in the next year, or the next five at the human level. It is a direction for research of a treatment for this disease. It is new, and very interesting. This is an important advancement that has taken place in the past year.

There is new information about the biochemical function of Frataxin, the protein that is deficient in FA patients. Frataxin is a mitochondrial protein that is localized in the structures that exist in each cell in our body and are necessary to produce energy. Frataxin, in ways that we don't understand, controls the flux of iron in and out of the mitochondria. If Frataxin is deficient you have too much iron.
The iron reacting with oxygen can generate toxic molecules, free radicals. Most of this knowledge comes from studies in yeast that has a Frataxin gene like we do.

Last year a number of studies have given evidence that the same mechanism can be occurring in the human disease by finding released mitochondrial iron in the cells from patients with FA and evidence of free radical damage. In addition to this study, there have been studies that suggest a biochemical function of Frataxin.
This was presented at the American Society of Human Genetics last October, by a group working at the Mayo Clinic. Their proposal is that Frataxin is a protein able to bind iron. If you take iron and put it in solution in a test tube, the iron will precipitate. It will be oxidized by the oxygen in the air and form an insoluble precipitate.
If there is Frataxin present in the same test tube and you add iron, the iron will stain the solution, but will not precipitate. There is preliminary evidence that Frataxin may be binding iron and preventing it from precipitating.
Therefore, Frataxin would have the function of preventing iron in mitochondria to react with oxygen and form toxic free radicals because it will bind it and isolate it from the surrounding environment. This was presented at an international meeting and it is still a work in progress. This is not final yet.

More progress was made when information came out a few days ago from a group in England, who has worked out the structure of Frataxin. What does this mean? A protein is a molecule that is made up of many amino acids like DNA is made by four possible units, A, C, G, and T, that follow each other in a long chain. Proteins are made by twenty different possible units called amino acids.
They are also arranged in a chain and the code to make a protein is contained in the DNA. The FA gene contains the code to make the protein called Frataxin. We know the sequence of amino acids of this protein because, by knowing the code we understand what the sequence is. Then, the protein chain does not stay in a long chain, it is not free floating in the environment, it folds on itself in a specific way giving the protein a specific shape which determines the function of the protein. Because of its shape it can interact with chemicals, promote certain chemical reactions, or participate in the formation of structures in the cells.

The investigator used a very refined technique to determine how the Frataxin molecule is folded and what shape it has. Frataxin is a globular protein and it has a surface that is highly conserved of Frataxin in all living things. A certain portion of the surface of the molecule is basically identical and this points out that this portion of the surface may interact with something that we have not yet identified, but is essentially the same thing in all organisms.
She also found that some of the point mutations, that rarely cause the disease, badly destabilize the structure making it unfold, preventing the protein from adopting its proper structure. This is an important finding that we are not yet ready to interpret, but it will provide important tools to work out the function of the protein.

There has also been an effort to develop a mouse model for FA to study the pathology and pathogenesis of the disease (how it develops and what kind of damage it causes). You can dissect an animal at different stages of development to see what happens to the body as the disease is developing and progressing. You can focus on specific tissues that are hard to get from patients, like the heart, and nervous system.
You can breed mice (it only takes about three weeks) so research makes faster progress. We can cross mice that have a specific variation of other genes, like iron metabolism and free radicals to work out what Frataxin is doing and what genes interact with the Frataxin gene. You can also test treatments in mice like drugs, gene replacement, gene therapy approaches, attempts to destabilize repeats and so on. It is very important to have a mouse model.

There isn't a natural mouse model for FA. The first attempt to make a mouse model for FA showed that a complete absence of Frataxin is lethal. Therefore, if there was a spontaneous mutation in the mouse that led to the disruption of Frataxin the mice would not develop.
The specific GAA expansion that we find in humans could not develop in the mouse because the starting sequence is not in the mouse. We have to make a mouse, we cannot find one. There are a number of problems when developing this model. We have to reproduce the defect as much as possible as we find it in patients. We have to create a deficiency, but not a complete lack of Frataxin and to reproduce the tissue distribution of the Frataxin deficiency.
We need to have mice that get sick within a reasonable time, and need to reproduce all of the main features of the human disease including, involvement of the nervous system, cardiopathy, and the risk of developing diabetes.

What kind of approaches can we follow? One is called the 'knock out,' developed by Dr. Michel Koenig; this is a way that you totally disrupt the gene. It has been done, and it leads to fatality. The mice don't develop. It is still an important model to have in the laboratory, these mice can still be used for a number of experiments.
For instance, we can try to give the pregnant mothers drugs or treatments to see if it helps with the development of the embryos that are defective in Frataxin. This is one of the grants that was funded by the NAF. This type of work is also going on in my laboratory. We are trying to treat the mothers with anti oxidants or an iron deficient diet and see if anything happens, if the development of the embryos changes in any way.
The other thing that we are doing is crossing mice with other mice that are deficient in genes that are necessary for the cells to kill themselves, to see if this changes anything in the lethality of the model.

In heterozygous mice, one copy of the gene is disrupted, and one is normal like the carriers of FA. They have 50% of the protein so you can do further manipulations on the mice to get the deficit that is substantial and leads to disease. You start with a 50% deficiency instead of 100%. This may be helpful to create a deficiency model.

We have been able to insert the GAA expansion in the mouse gene in the same position that it is found in the human gene. This work has been done through a close collaboration with Jerry Kaplan at the University of Utah, and is a joint effort between his lab and mine.
We know that the repeats are stable in the mouse. We have been able to insert a small repeat in the gene which is important because we have obtained viable mice that are homozygous with two repeats. Theyhave been born, and have started growing but are still very young so it is too early to tell if they are sick or not. We have crossed this mouse with a knock out so we have mice that have a GAA repeat on one chromosome and the gene is disrupted on the other.
We are doing pathology studies to see if there are possible developmental problems in these animals that would suggest the same thing occurring in FA.

We suspect that part of the problem in FA happens very early and possibly during development, in particular the loss of the sensory fibers in the nerves. We are doing this type of study in the mice as an initial characterization because it would tell us we are on the right track to developing the mouse model.
We are also doing studies on the effect of Frataxin deficiency on the differentiation of cells that become neurons. We have evidence that cells that are Frataxin deficient may die while they try to differentiate and become neurons. This may be the basis for the loss of sensory neurons in patients with FA which would cause sensory neuropathy.

So, now the thing that everyone wants to hear about....the treatment. The attempts are based on the fact that we assume that the problem is due to the production of free radicals because of excessive iron in the mitochondria. There are a number of drugs that can, in theory, be utilized for this purpose.
One can use a drug to remove iron from the system, and in yeast it is suggested that this may work. Another important set of findings is that when yeast cells, deficient in Frataxin, are grown without iron they are fine.
If you add iron, then, they start to have abnormalities. This suggests that the problems in FA may be iron dependant, but humans are not yeast and we do not know if you can take iron out of the human system without killing them or creating other problems. We can take enough iron out of the system to prevent the problems dueto Frataxin deficiency.
An attempt has been made at the University of Utah to treat a few patients with an iron chelating drug. The patients are given enough of the drug to start inducing anemia. They have been evaluated over a year, mostly for cardiac problems. We are eager to know the results of the study. It is still being evaluated, but we should know something in the next few months.

Other drugs that can be used are anti oxidants. There is a French group that has proposed usinga substance that resembles co-enzyme Q. Co-enzyme Q is normally found and is active in mitochondria, and it has something to do with the respiratory process.
This substance can activate mitochondrial function and be an anti oxidant at the same time. It has been shown in the test tube to be protective against some of the damage that iron causes to tissue extracts. It has been given to patients and preliminary data suggests that it may have some efficacy, particularly for controlling the heart disease for FA patients.
It is very uncertain if it helps with the ataxia. This is why we are trying to set up studies to answer this question. Pilot studies with co-enzyme Q, or idebedone are going on in a number of centers, including Montreal. These are very preliminary pilot studies to assess if the drug has any problems of toxicity in patients with FA, and if we can get an initial hint of whether it will be effective or not.

Maybe next year there will be some results that will further clarify if these drugs really have use as far as treatment of the disease.
Hopefully, we will move on and add drugs to the treatment and see if we can improve the situation. Many exciting things are happening and soon we will have animal models and results of the clinical trails. We will be able to say that we have entered a new phase in the study, and hopefully in the management of the disease.