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Scientists don’t know what causes Amyotrophic Lateral Sclerosis (ALS). But many do believe, with relative certainty, that there are six biological “steps” that must occur in order for the onset of the disease to occur.

It may seem counter-intuitive at first, but by analyzing population data about what age ALS onset tends to occur researchers have determined that there are six distinct biological processes that must occur to precipitate ALS. Additionally, they have theorized that some ALS-related genetic mutations, such as SOD1, TDP-43, and C9orf72, may each account for a certain number of steps – for example, an SOD1 mutation may account for four of the six steps required to cause ALS in a person without a genetic mutation.

Ammar Al-Chalabi is a Professor of Neurology and Complex Disease Genetics at the Maurice Wohl Clinical Neuroscience Institute at King's College, London. Dr. Chalabi was an author on the studies that first introduced these theories in ALS. He spoke with the ALS Therapy Development Institute (ALS TDI) to explain how we can determine the number of steps required to cause a disease, even without knowing what the steps are, and what this might mean for research into treatments.

**What does it mean when we say that the onset of a disease is a “multi-step process”?**

So, what that means is, instead of there being one trigger for a disease, there's actually a set of triggers that all have to line up. For example, you might think "I carry this gene, therefore I'm going to get ALS,” or “I've smoked, therefore I'm going to get lung cancer.” It's not like that.

If we take the smoking and lung cancer example, a multi-step process means that smoking on its own isn't enough to give you lung cancer. You have to smoke for a particular length of time. And during that length of time, you're exposed to various toxins that affect your lungs. Lots of different changes happen, and as each of those steps occurs, eventually the cancer is triggered.

In ALS, it's probably something similar. **So, for example, you have some level of genetic burden, but you're not going to get ALS just by carrying that genetic burden.** You need some other triggers. You need some other risk factors. Let's say heavy exercise is one of those other factors. Doing heavy exercise on its own isn't going to be enough to give you ALS. You need to have the genetic background and the heavy exercise and whatever other steps are needed. **We know from using some statistical tools that there are six steps that you need to go through to get ALS, six steps that happen inside the body.**

**Can you clarify what a “step” is in this context?**

**A step is a biological change in one cell of your body.** It probably happens in lots of cells in your body. But, eventually one of the cells goes through all the six steps and finally triggers ALS. That likely then leads to a cascade of problems that spreads throughout the body.

As an example, we do know that **different genetic mutations will predispose you to a certain number of steps.** SOD1 gives you four steps. TARDBP accounts for two steps. C9orf72 accounts for three steps.

**So where are these numbers coming from? How do we know that there are 6 steps for most cases of ALS?**

It's an idea from a couple of people from the 1950s, Peter Armitage and Richard Doll, who worked on the mathematics of cancer, and they developed this idea of the multi-step hypothesis. It's relatively simple to follow if you imagine that, let's say, drinking tea reliably causes ALS. You could work out the incidence of ALS by looking at the incidence of tea drinkers because everybody who drinks tea is going to get ALS.

It obviously isn't true, but let's say that it was. Now let's say, actually, it's not enough to drink tea. You have to have a step beforehand. So, you have to drink tea and do something else. Let's say it's eating mustard. In this example, the probability of you eating mustard over the course of your life has to be multiplied by the probability of you drinking tea, and that will be proportional to the number of people who get ALS in the country.

That's the basic idea of the math. You can just do that for as many steps as it takes. **Now, if you follow that math to its logical conclusion, you can end up with an equation in which there is a relationship between the age of onset of the disease, the incidence of the disease at each age, and the number of steps.** It's just by taking that first idea the risk of having being exposed to whatever the risk factors are. That gives you, in the end, some math that shows that it's a straight-line relationship between the log age of onset of the condition and log incidence, which gives you the number of steps.

So, for our study, we needed information about the incidence of ALS at different ages. Because if you have that information, then, you can work out how many steps are needed. We have a population register in the UK called the SEALS Register, the Southeast ALS register. It's in the southeast of England. And because that's a population register, I was able to use that to work out the incidence of ALS at different ages and then take the log of the age and the log of the incidence. Make the straight-line graph and look at the gradient, and the gradient is one less than the number of steps.

**I could see the gradient was five, and therefore it's a six-step process.** I went to my colleagues in Scotland and did it with their registry, and we got exactly the same result. We went to our colleagues in the Netherlands, Ireland, and Italy and we got exactly the same result again with all of them. And if you combine them, they also give you the same results. So, it tells you they've all got exactly the same slope, which is five, and therefore it's a six-step process. Since then, others have done this in Australia, Japan, and Denmark with the same result, and a 5-step result in South Korea.

**So then how do you know how many steps a certain genetic mutation might account for?**

You can just look at the people with one genetic variation in your population registry. And you look at the number of steps they need. So that's what we did in Italy, because they've got good population register data with genetic information, so they could show that people with these different gene variations have these different slopes.

**What are the implications of knowing the number of steps behind ALS for research into treatments and drug discovery?**

The way you can work out what these steps might be is, for example, you can look just at the people with a genetic variation in your population register. And you look at the number of steps they need. You can just pull out the people with those risk factors in your population and then look at the incidence of ALS in that group.

If you can do this on a large enough scale so that you've got enough information about your population that you can ask repeatedly, "well, what about this risk factor? What about that risk factor?" Y**ou might be able to find a pathway that doesn’t have to do with genetics, or that interacts with genetics in some way.** And that just gives you another insight into what could work as a treatment.

At the moment, all of our treatments are really based on either genetic understanding or on pathology, looking down the microscope of the cells and saying, "well, this protein seems to have gone wrong. Let's make a treatment that affects that."

For example, if you found that smoking caused ALS. Actually, we know pretty well that it doesn't. But let's say it did, then you could further investigate, how does it do that? You could look at which toxins are doing what, see what pathways triggered the cell death, and make a treatment that affects them.

**What to Do Next**

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