How inbred dogs help us learn about human diseases

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How inbred dogs help us learn about human diseases

Dogs are helping scientists figure out why our copper storage systems can go haywire

Though dog slobber can be gross, it turns out the DNA in your dog's kisses is actually a treasure trove of genetic evidence that could help scientists determine and treat the causes of diseases in dogs – and humans.

Human bodies – and in fact, the bodies of all animals, including dogs – need copper. That's because many proteins require copper to function properly. The metal plays many important roles, including helping to absorb iron, allowing neurons to communicate, and protecting cells from highly reactive "free radicals" of oxygen. But we don’t actually need very much of it, and excess copper can be dangerous. The little copper we do require needs to be carefully regulated; the same highly reactive properties that make it great for increasing chemical reactions we do want also allows copper to undergo reactions we don't want. To maintain the fine balance we need, our bodies have developed copper regulation down to a science.

We ingest copper all the time from the food we eat, including whole grains, beans, shellfish, and dark leafy greens. To use copper effectively, first it needs to enter the bloodstream. Copper enters cells in the intestines called enterocytes, where a copper transporter, a protein called ATP7A, pumps it into the bloodstream. From there, blood goes to the liver to "detox," where things that can be dangerous, like excess copper, are removed or neutralized. When copper levels are too high, a related protein called ATP7B—found in liver cells known as hepatocytes—pumps copper from the liver into the bile duct, so it can be disposed of in our feces.

These two copper transporters look a lot alike and work similarly, but because they’re expressed in different locations, they have very different functions – and consequences if they don’t work. Mutations in ATP7A cause a fatal disease of copper deficiency called Menkes disease (MD). The enterocytes of MD patients take in copper, but they can't pump it out, so they get overloaded and slough off. Without these enterocytes, copper can't be absorbed out of food and delivered to all the cells around the body that need it. So those cells start to malfunction too, leading to curly sparse hair, stunted growth, progressively severe neurological problems, and ultimately, death.

Conversely, mutations in ATP7B cause a disease called Wilson’s disease (WD). Because their ATP7A protein is intact, these patients are able to absorb copper into the bloodstream, but their liver cells can't get rid of enough excess copper, so it starts to accumulate, eventually causing liver problems. Additionally, though scientists aren't entirely sure why, copper also often starts to build up in the eyes – causing characteristic golden rings around the cornea called Kayser-Fleicsher rings – and the brain, causing neurological and psychological symptoms.

While scientists know what causes Wilson’s disease, some patients have similar symptoms but no known protein mutation (a condition known as idiopathic copper toxicosis). Additionally, even in patients with identified mutations, the age of onset and the severity of symptoms can differ greatly. For example, some patients with WD only have liver problems, whereas others also have neurological symptoms. Some of these differences can be attributed to where in the protein the mutation occurs—there are close to 300 known WD mutations, and these different mutations may alter the protein's function in different ways—but much of the variance is still unaccounted for.

Some of why the same genetic disease can have different symptoms in different people comes from environmental factors, such as what we eat, how much we exercise, and what we're exposed to. But in addition to environmental modifiers, disease severity can be affected by genetic modifiers—essentially differences in other genes can either protect us from some of the disease's effects, or make the symptoms worse. This variety can make it hard to identify a causative gene, let alone any modifiers, especially when you have a rare disease with a small number of patients and many types of symptoms.

So when scientists want to find the genetic roots of a heritable disease, they commonly do an experiment called a genome wide association study (GWAS). In a GWAS, researchers take DNA samples from a large number of people, some who have the disease and others who don't. It would be too expensive and computationally intensive to do traditional sequencing, where they would have to read out every base in the genome. Instead, for each sample scientists check the genetic sequence at locations known to vary frequently. These "single nucleotide polymorphisms" (SNPs) aren't mutations per se (although all genetic variation starts from mutation), but rather natural variations, similar to how people have different hair colors.

Scientists typically sequence tens of thousands of SNPs spaced out along the genome, splitting the chromosome up into segments. They can then compare between people who have the disease and ones who don't. Over most of the genome, there won't be any correlation between the two groups, but around a causative gene, the groups should look very different, because that gene (and the DNA near it that was inherited with it) will be different.

The GWAS has a limited resolution because the initial regions scientists identify – called linkage disequilibrium or "LD blocks" – are quite large. In general, the size of theses blocks depends on how far apart the SNPs are, and how many consecutive SNPs are included. This means the LD blocks often contain multiple genes. Once scientists identify a large region where the DNA of people with the disease differs from that of people without the disease, they need to dive in deeper, "fine-mapping," to see what gene (or sometimes multiple genes) might be involved. Depending on the size of the LD block, they might sequence the entire region, or just the parts they think could be important, this time analyzing each nucleotide.

This process can help researchers identify "candidate genes," or potentially dysfunctional genes causing a disease, but there's actually no guarantee there is a direct correlation. For instance, often in addition to inheriting a disease people inherit a lot of other things that may look genetically similar, but have nothing to do with the disease itself. This is a problem called population stratification.

Inbreeding for the win

To get around this problem in a laboratory setting, scientists often perform backcrosses, or inbreeding of populations of animals or plants to reduce the background genetic variability – you can think of this as genetic "noise." As variability elsewhere decreases, the causal region will start to stick out more. 

Obviously we can't do this with humans, but thankfully we have dogs! There are many problems with inbreeding – genetic diversity is important for allowing species to adapt, as well as preventing recessive diseases, which require two copies of a faulty gene – but the long history of inbreeding in purebred dogs makes them excellent resources of DNA for studies. Dogs within the same breed have low overall genetic variation, meaning the faulty regions will stick out more, as well as large LD blocks, meaning scientists don't have to sequence as many SNPS.

The first dog to have her genome sequenced was a boxer terrier in 2005. Since then, new tools have been developed to streamline the analysis and comparison of canine DNA. The results have unraveled a few mysteries of evolution, such as the inheritance of complex traits, like body size, as well as the causes of a variety of genetic diseases. Given these promising results, researchers wondered if dogs might also hold the key to understanding human copper storage diseases.

People have known for a long time that dogs can suffer from similar copper storage diseases. In the early 2000s, researchers found that in one particular breed, Bedlington terriers, an excess copper disorder was caused by deletion of the COMMD1 gene. (The research actually led to the discovery of a whole new family of proteins.) But most humans with WD had mutations in a different gene, ATP7B. So scientists at several universities, including Utrecht University in the Netherlands and the University of Missouri, decided to collaborate to see if similar diseases in a different dog breed had different genetic causes, hoping to find an animal model that better matched the human disease. So they turned to the Labrador retriever.

The researchers collected DNA from almost 300 Labrador retrievers, looking for regions of DNA that differed between healthy dogs and dogs with copper toxicosis. They found LD blocks encompassing ATP7A & ATP7B, and when they sequenced these regions, they found that dogs with copper toxicosis had a mutation in ATP7B—but their symptoms were often less severe if they also had a mutation or variant in ATP7A.

Once they identified these mutations, the scientists needed to prove that they really did have an effect on copper processing. Next, they expressed those mutant proteins in cells and looked at how the mutations affected the proteins' ability to transport copper and move throughout cells. It turns out the ATP7B mutation caused copper to accumulate in liver cells, like in human patients with WD. But the ATP7A mutation caused copper to accumulate in cells similar to enterocytes. So in dogs with both mutations, the researchers think this reduces the total amount of copper reaching the liver, helping it avoid becoming overloaded, and thereby lessening the disease's severity. In people, this raises the possibility that variants of ATP7A might also have a protective role, helping explain some of the variability in disease severity. A previous small study didn't find any cases of ATP7A variation impacting the disease course of WD patients, but clearly more research is needed.

The copper connection

It turns out Labradors and humans with too much copper have a lot in common: in addition to having mutations in the same gene, they develop a lot of the same symptoms: both accumulate large amounts of copper in the liver, which can lead to liver cirrhosis. But there are also differences: in human patients, copper doesn't only accumulate in the liver – it also builds up in the eyes and the brain. 

Another potentially important difference is the role diet can play. In Labrador retrievers, low-copper, high-zinc diets can sometimes help dogs avoid the need for chelation, a medical treatment to remove heavy metals from the body. In people with WD, altering diets to avoid copper doesn't help much, but it can help patients with copper storage diseases whose causes are still unclear. It's possible that more genetic modifiers of copper processing remain to be found in Labradors, which could help us identify more causes and modifiers in humans, potentially leading to more effective treatments.

Research on dogs could also help patients (of both the human and canine varieties) who have symptoms like copper disorders, who don't appear to have a known protein mutation. It’s possible that in dog breeds scientists haven't examined yet, researchers could identify more causative mutations and genetic modifiers. Even in the dog breeds we have studied, there remain mysteries to be solved. For example, while COMMD1 deletion can account for most cases of copper toxicosis in Bedlington terriers, some Bedlingtons with the disease don't have the mutation—suggesting another gene might be involved.

In addition to helping us find genes that may cause human diseases, dogs can serve as "model organisms," helping explain how specific genes cause disease, as well as developing effective treatments for us and them. Humans are more closely related to mice than dogs on an evolutionary time scale, but mice, with their shorter lifespans and faster breeding, have experienced a faster rate of genetic evolution, so their genomes are actually less similar to ours than dogs' are. Because of dogs' larger size, longer lifespan, and similar environmental exposures, their disease courses and responses to treatment often better match those of humans.

Nor is it a one-way street: By better understanding the similarities in these disorders, treatment for dogs can also be improved. Even if the underlying causes are different, diseases with similar symptoms often respond to similar treatment. Because of of the similarities in copper storage diseases in humans and dogs, they have typically received the same treatment: copper chelation, usually with the medication d-penacillamine. ("Chelate" comes from the word for claw, because these drugs grab onto metals like copper and cause them to be peed out.) But d-penicillamine is a harsh drug, and can have serious side effects, including the worsening of neurological symptoms. A lot of research has been conducted in the search for better alternatives for humans. But partly because dogs don't show the neurological symptoms that these newer drugs target, they haven't been tested much on dogs. Now, one of these drugs, ammonium tetrathiomolybdate (TTM), used for several years in humans, is being tested in a clinical trial for dogs. A group of researchers at Michigan State University conducted a pre-clinical study which showed that TTM was safe in dogs, and they recently completed a clinical trial (the results aren't out yet).

This is just one example of the “One Health” approach, which encourages collaboration and communication between veterinary medicine and biomedical research, with an emphasis on the interconnected nature of the health of humans, other animals, and our shared environments. After all, all animals get sick – and by studying the similarities and differences in these illnesses, we can improve treatment for everyone.