Late Onset Cerebellar Ataxia: clinical & genetic characteristics

Transcription of Isabelle Thiffault’s speech at the General Assembly of CAFA on November 4, 2007

Diaporama PowerPoint (in French)

Hello, my name is Isabelle Thiffault, I have been doing a doctorate for three years and a half under the supervision of Dr. Bernard Brais. During the first years of my doctorate, I was an CAFA grantee. Now I have a government scholarship but we also receive allocations from your Foundation, which helps us starting new projects.

Unfortunately, Dr. Brais is at the hospital and cannot be with us today. That’s what happens with the shortage of neurologists we all know. I will try to do my best to represent Dr. Brais. Miriam Srour is also working with me, she’s a new neurologist specialized in paediatrics and is undergoing an apprenticeship at our lab. She worked a lot with me on the project of which I will give a clinical description here. She has also worked with me on other forms of muscular diseases that are not necessarily ataxic. She would have liked to be here today but unfortunately she has to stay alongside Dr Brais.

As mentioned before, last year we were studying a form of ataxia called the Beauce ataxia. We have already started to gather new patients with forms of ataxia of which we ignored the cause. As of 2005, we started to form a new group of patients with what is now called the late onset ataxia. Of course, the name implies that it’s an ataxia that develops very late. Most of our patients develop it around 60 unlike the ataxias that you know very well, such as Friedreich’s ataxia and Charlevoix-Saguenay ataxia, which are slightly more severe forms, sometimes much more severe, that appear at a very young age. The Late Onset Cerebellar Ataxia (LOCA) is a degeneration in the rear part of the brain, called the cerebellum. From now on I will use the term LOCA.

As you can see on the figure I’m showing, there is a huge amount of projects in the neurogenetic lab. Far too much for the number of people who work there. We are also working on muscular dystrophy, congenital myotonia, ataxias of the Gaspésie region (there are two of them: the AOA2 ataxia and a polyneuritis, which is also a recessive ataxia and leukoencephalopathia, my first project, formely known as the Portneuf ataxia), sensitive neuritis in Lanaudière, limb-girdle dystrophy, congenital dystrophy in the Montreal region, Beauce ataxia and now we can add the late onset ataxia, which I will present you today.

It is important to note that even in small regions like Saguenay-Lac-Saint-Jean there are often mutations of congenital ataxias. For example, there are five mutations of the congenital myotonia in Saguenay-Lac-Saint-Jean. You can see now how complicated it is. As we discover more diseases, we realise that they are being discovered now because there are more mutations in a more heterogeneous population. I will come back to this later.

The founder effect is one of the research hypotheses about the mutations found in Quebec. We believe that up to four mutations have been implanted among the Quebec population. They have possibly arrived during the formation of Quebec, but could also have been brought from France or Great Britain, depending on the origins of our patients.

In a pedigree, we are looking at the mode of inheritance and like in most ataxias you know it is the autosomal recessive mode of inheritance. This means that in order to get the disease, both parents have to be carriers of two gene mutations and the patient has to receive two copies, i. e. a copy of the gene mutation from the father and the mother. The ataxias we are discovering today are complex because their phenotypic variation is very large. Even in a family with three affected members, i. e. brothers and sisters, we see a huge difference in phenotypes. In some the disease starts out early but develops very slowly and progressively. In others the disease starts later but progresses very fast. I put up a little graph to show you what a founding mutation is. As I said, the mutation could have come from Europe or have happened here in Quebec. There is a mutation that occurs in a germ cell. More often mutations occur in our sexual cells, i.e. in spermatozoids and ova. This mutation will transfer to the child. If the mutation occurs during this event, the child will not get the disease. If the foetus doesn’t die because of the mutation, the person will become its carrier. Later, if the person has children he could transmit the mutation to them. This is why there is a hereditary process. A person develops the disease because there were two carriers who didn’t have the disease, but whose child received both alleles with the mutation. It happens at the parents’ level because genetic combinations and mutations are frequent. They are transmitted to the children who transmit it to their children and then it spreads in the population. You can see on this graph that both parents are in blue, they don’t have the disease. M is the gene with the mutation. Both parents are carriers. Here the child received two chromosomes with the mutated allele, so he or she has the disease (in red). If we read the DNA sequences, the control being blue, the parents have one red allele and one blue allele, while the child has only the red allele. This is to show you what we look at in genetics.

The first objective of our research project was to finish recruiting patients. In reality the recruitment of patients can take years. The first patients were recruited in 2004-2005 and we only finished in 2007. Obviously the clinical description of the disease is also important. You realise that the different forms of ataxias are multiplying. We need to tell doctors what are the particular symptoms of this form of ataxia so that they are able to diagnose it. First of all we always need to reject the known forms of ataxias.

I will now present the material and the methods that we used. As I said the recruitment started in 2005. We thought that it was a hereditary form because in the first family that we saw, three people were affected. When we started to question the children, the cousins, uncles and aunts we realized that the parents came from a small village in Quebec.

However there was no consanguinity as they were not first cousins. It was probably a hereditary and recessive form of ataxia. We went to Saguenay and we started to recruit families with very similar forms. Saguenay-Lac-Saint-Jean is known for genetic founder effects. It’s not that there are more genetic diseases in Saguenay but there is more homogeneity because of how it was formed. It’s more concentrated because people who were born in Saguenay tend to stay there. There is not much exchange with the rest of Quebec. Now we know Saguenay better than other regions and we often hear about forms of ataxias in children, so we concentrate our research on them.

Here is a Quebec map. It shows the geographic distribution of our patients’ origins. Note the two white circles that we have drawn. In fact there are a lot of families from Saguenay-Lac-Saint-Jean and the Eastern Townships. In regions like the Eastern Townships, there are French Canadians but also many families with English names. The genetic mix is much more significant and it makes our genetic analyses harder.

This schema shows that there are over 35 families now and we have 183 different DNA samples. We recruited patients from all over Quebec in order to get the most genetic information possible.

It’s always complicated to work on late onset diseases. Most patients were diagnosed at 60 and their parents were often dead, probably also their siblings. Therefore it is hard to obtain a lot of study subjects. The more people we have in a family, the more chances we have to find the gene and its mutations. Still, you can see that we did a good job as we were able to get a lot of patients and families in two years.

This table shows the characteristics of this form of ataxia to patients and doctors. In average this disease develops at 60, but there are patients who have developed it in their 50s (52-53 at the youngest). However some were diagnosed at 88. Often the disease develops but it takes years before the patient sees a neurologist who will diagnose it. As you can see, the first neurological exam is done at 88. There are from 15 to 20 years between the first symptoms and the diagnosis. 48% of our patients need a cane or a walking aid. 29% are in a wheelchair. This form is therefore much less severe than others that you know, such as the Friedreich ataxia and the Charlevoix-Saguenay ataxia, when even children are in wheelchairs.

As for other symptoms, there are two Quebec regions with the highest number of families: Saguenay-Lac-Saint-Jean and the Eastern Townships. There are as much men as women affected, so it’s autosomal, which means that it’s not linked to the sex chromosomes X and Y. It’s a recessive form, which means that the parents are not affected. The main clinical characteristics are the presence of ataxia in all cases and dysarthria in 81% of cases. With brain imagery we see cerebellar atrophy in 88% of cases and frontal lobe atrophy in 50% of cases. 52% of our patients have nystagmus, an ocular phenotype. However we can’t be sure if it’s typical of the disease or if it’s caused by the aging process of patients who are 60 years old and older. 29% of patients are in a wheelchair. When we exclude all other forms of ataxia, we always start with the known ataxias.

Obviously we tested for Friedreich ataxia and type 2 and type 6 spinocerebellal ataxias. We have also tested for X fragile syndrome and for the two other forms of ataxia that we told you about, Beauce ataxia from last year. We studied haplotypes and mutations. I think that we presented you two mutations last year. Today, in 2007 we are at seven mutations for the Beauce ataxia in the Quebec region alone. My main project is the Portneuf ataxia (ARSAL). I have also excluded these two loci for all patients. Therefore all the families are obviously negative for these forms of ataxia. We didn’t find any mutation, any homozygosis or allele sharing in these regions. This makes it a new form of ataxia.

The second step is to try finding the gene responsible for this disease in the genome. We use microsatellite markers provided by an Icelandic company. We test 388 markers everywhere in the genome. These studies are fairly expensive: the first step alone costs about $200 per patient. Then we need to do a genetic mapping: when we identify the region on the chromosome we need to find a gene in that region that causes the disease. We analyse all the genes of the region by sequencing, like the small coloured spikes that I showed you.

To do these genetic studies the families need to be large enough. I told you that we have 35 families with at least one member suffering from LOCA. We recruited everybody, 188 DNA samples in total, but not all families have enough members for genetic analyses. We did the studies anyway but we only took the larger families with the largest number of patients for the first step of the research.

As I said, we use microsatellite markers, small repetitions in the genome called CA. We test them everywhere to try and see the difference between individuals who have the disease and those who don’t. We then need to analyse 388 markers. Now there is a software that allows us to identify the region responsible for this disease. These are linkage values called LOD. (I’m not good enough in computers to explain how this software works). This is how we will find the region in the chromosome.

This graph was made by this software. It gets more positive as it gets higher. The gene is between the markers. This is how we find out where is the disease in the genome. Then we compare the chromosomes of all the families. I always make color codes. You can see that there are a lot of green chromosomes – it’s our Saguenay chromosome. The orange chromosome is for Eastern Townships anglophones. You can see that some patients have both chromosomes. It means that their parents come from Saguenay and the Eastern Townships and were both carriers of the disease.

Here you see the DNA molecules and CA repetitions that I told you about. Here is a gene, a small bit of DNA. It’s the DNA molecule. It forms chromosomes that are in the core of our cells in our entire body. Thus, in a genetic hereditary disease, mutations occur not only in the brain cells but all our body cells carry this affected allele. However there are certain regions, tissues and organs where the disease develops.

The LOCA locus is located on chromosome 13. Just to remind you, there are 23 pairs of chromosomes or 46 chromosomes in total in the human genome. Here we have our two chromosomes 13, there we have chromosomes 1 to 22 and the sex chromosomes. The disease I am presenting is located on chromosome 13.

Now, once we have marked the upper area, I could locate the disease between markers. Then I can look on a map (there are lots of databases on Internet) to see which genes located in that area could have caused the disease. This area is approximately 4 centimorgans (cM) long. It is still very large: it contains 25 genes. This means we had to analyse 25 genes. This is a lot of work and it’s very expensive – sequencing each gene costs thousands of dollars. We have decided to use a new technology similar to the one I described earlier and involving repetitions but with a slight change. It’s not a CA repetition; it’s just a letter, for instance T, that we change for another. It allows us to tests them with a technology called DNA microchips. The DNA microchip is a small glass plate. On the surface each plate contains thousands of synthetic DNA sequences. We insert the patient’s DNA in these plates and the results are analysed by a machine. Then we analyse each person with lasers, it’s really high-tech. We try to see if we can get closer to the gene that causes the disease. This technology allowed us to confirm that the disease we describe is located on chromosome 13. We managed to confirm it and to reduce the distance. The previous method we used with deCODE gave us 388 markers, while this one allows us to test over 350 000 markers. This is why we need people with good computer skills to analyse this huge amount of data. We analyse 350 000 markers for each patient. This is preliminary data because instead of the 4 centimorgan-long area that contains 25 genes we have a small 0.5 cM-long area. It’s a huge difference. I will confirm the results in the next few weeks. This means that instead of sequencing 25 genes, we still have 2 or 3 to analyze. We didn’t have access to this technology before. It has been used in Montreal for a few years now, at the McGill University Genome Centre, but it was too expensive for us; testing one person used to cost a thousand dollars. But a year ago the price went down and now it costs $200-$250. We can afford to use it to find the causes of genetic diseases.

In conclusion, LOCA has two founder effects: one is in Saguenay, the other is in the Eastern Townships. With the help of different centres, clinics and all of our colleagues (Dr Bernard Brais is working with neurologists from all over Quebec) we have recruited 35 families and collected 188 DNA samples. It’s the world’s largest LOCA cohort. Last week I presented these same results in California. We had a nice exchange with scientists from the United States and Europe. There were even people from Japan. They were interested in this project because it’s the first time we can prove that a late onset disease can be genetic and hereditary and not only be due to the aging process. We are very proud of this project. It’s important to emphasise that it is thanks to your support that we are able to carry out this kind of research.

I already mentioned that as we can see with our cohort, phenotype and severity are very variable. This is why we continue genetic mapping and we succeeded in demonstrating that the disease is located on chromosome 13. Then, thanks to the DNA microchips we have found that it’s a small area. Next year I will probably be able to show you the mutations and the gene.

When we analyse haplotypes we mark the two chromosomes orange and green. It explains about 77% of cases. For instance in many forms of ataxia there are at least 3-4 mutations in the gene that causes the disease.

We haven’t yet started the sequence analyses because we were waiting for the last results. Instead of analyzing 25 genes we will only have 2 or 3.

Because the disease is so severe we can suppose a triplet repeat. I think you know that as in the case of Friedreich ataxia it is the GAA area that is exponentially repeated. Although I haven’t seen any yet, we can think that it is the same type of mutation in this case.

It is important to note that it’s the first time we identify a locus for a late onset form of ataxia. Some cases even lead to multiple system atrophy. Dr Brais would have explained it better, but I will try to simplify what it is. Multiple system atrophy is more commonly known as Parkinson’s. A lot of our 80 years old ataxic patients have symptoms that resemble more Parkinson’s than ataxia. It’s interesting because Parkinson’s is related to the aging process but there could be more genetics than we think. Maybe even ataxia could be the first phenotype that we could observe in some patients before they have Parkinson’s. This means that we could use medications normally used for Parkinson’s. Maybe even before the development of the Parkinson syndrome, but we’re not there yet. These are only some of our hypotheses.

My short term goals for this project are to finish analyzing DNA with the microchips, sequencing the candidate genes of the area and find the responsible mutations. In the long term we want to sequence all our patients who have developed MSA for that genome and this could help us find the causes of Parkinson’s in other people. Once we find the mutations and the causes we will be able to offer a diagnostic test as well as a treatment to patients. In order to do the treatment we need to understand the protein, so we will also analyse the protein to understand its role in the brain, why does it cause ataxia and why does it end up causing Parkinson’s.

Then, just as we have started to do with other projects such as Charlevoix-Saguenay ataxia we will probably make an animal model, such as a transgenic mouse model. Animal models help us to test drugs and to better understand the disease. We also have a fly model that we use to study a form of ataxia. We use flies because even little developed organisms such as flies have important genes, especially in the brain. It helps us to better understand the protein and to quickly test molecules to see if we can obtain a treatment.

First I would like to thank all the families participating in the study. I also would like to thank the Claude St-Jean Foundation because we can’t start projects like that without the support of a foundation such as yours. For many years now the foundation provides great support for Dr. Brais and provides grants to the students who work on ataxias. We are very proud to present the CAFA logo each time we make a presentation abroad. I hope that your Association’s influence will grow more and more. Thank you.