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This document is made available by the Howard Hughes Medical Institute.

Meet Tasha, a boxer dog (Figure 1). In 2005, scientists used Tasha’s DNA to obtain the first complete dog genome sequence, which contains 2.4 billion pairs of nucleotides over 39 pairs of chromosomes! Genome sequence data like this is useful for many reasons. In this activity, you’ll see how this data is used to find genes associated with different traits, such as fur color in dogs. The approach you’ll learn about is called a genome-wide association study (GWAS), and it can be used to learn about the genes of any organism, not just dogs.

PART 1: Introduction to GWAS

Read the information below to learn more about GWAS and its applications, then answer the questions that follow.

What Is GWAS?

GWAS is a method for identifying the genes associated with an organism’s collection of traits, or phenotype. It involves searching the genomes of many individuals — for example, many different dogs — to find DNA differences, or variations, associated with particular traits and phenotypes. After sequencing Tasha’s genome, for example, scientists sequenced and compared the genomes of many dogs from a variety of breeds. They found millions of common variations among these genomes. Some of these variations were associated with the color, length, or texture of a dog’s fur. You’ll learn more about these variations later in this activity.

1. In general, why do you think GWAS is useful? What kinds of problems could GWAS be used to solve?

What Are SNPs?

The variations found in GWAS are usually common variations in DNA sequences called single nucleotide polymorphisms, or SNPs (pronounced “snips”). A SNP is a variation in a single nucleotide, at a particular position in the genome, that occurs in over 1% of the population. Different variations of a SNP may be called SNP variants or alleles. Figure 2 shows two alleles for a common SNP in dogs.

As shown in Figure 2, SNPs are labeled based on the chromosome they are found on (e.g., chromosome 37, which is abbreviated as chr37) and their “nucleotide position” (e.g., 25,734,258), which is determined by counting from one end of the chromosome to the other. For the SNP shown in Figure 2, dogs may have either a C or an A. These two versions of the SNP are called the C allele and the A allele.

1. List the three combinations of alleles (C and A) that a dog could have for the SNP shown in Figure 2.

The locations of the SNPs in a genome serve as “markers “or “signposts” for the locations of genes associated with particular traits. A GWAS uses associations between a particular SNP and a trait of interest to predict how close that SNP is to a gene responsible for that trait. How does this work? In general, the closer two DNA sequences are to one another on the same chromosome, the more likely they are to be inherited together. So, if a SNP is close to a gene, it is likely to be inherited with that gene — and will thus be associated with the gene’s trait.

One way to find SNPs associated with a certain trait is by comparing groups with different versions of that trait. In a GWAS looking for genes that affect dog fur color, for example, we could compare the SNPs of two groups: dogs with black fur and dogs with white fur. We would then determine which SNPs are significantly more common in dogs with black fur compared to dogs with white fur. These SNPs are “markers” for regions of the dog genome that contain genes affecting fur color.

1. Why do you think SNPs are referred to as “markers” or “signposts”?

Figure 3 shows several possibilities for why a SNP is associated with a certain trait. The SNP may be in the gene that causes the trait or in a regulatory area for that gene. If so, the SNP could directly affect the gene’s function and the resulting trait. However, some SNPs in or near a gene may have no effect on the gene or its trait.

1. Consider the different types of SNPs shown in Figure 3: associated, unassociated, and causative (including both noncoding and coding).

   1. Which types of SNPs affect protein production or function for the gene of interest?

   2. Which types of SNPs might be identified in a GWAS?

GWAS in the News

Read the following news release, which describes a GWAS study with dogs. Note that a dog’s coat refers to its fur or hair.

Variants in just three genes acting in different combinations account for the wide range of coat textures seen in dogs — from the poodle’s tight curls to the beagle’s stick-straight fur. A team led by researchers from the National Human Genome Research Institute (NHGRI), part of the National Institutes of Health, reports these findings today in the advance online issue of the journal Science.

“This study is an elegant example of using genomic techniques to unravel the genetic basis of biological diversity,” said NHGRI Scientific Director Eric Green, M.D., Ph.D. “Genomics continues to gain new insights from the amazing morphological differences seen across the canine species, including many that give clues about human biology and disease.”

Until now, relatively little was known about the genes influencing the length, growth pattern and texture of the coats of dogs. The researchers performed a genome-wide scan of specific signposts of DNA variation, called single nucleotide polymorphisms, in 1,000 individual dogs representing 80 breeds. These data were compared with descriptions of various coat types. Three distinct genetic variants emerged to explain, in combination, virtually all dog hair types.

“What’s important for human health is the way we found the genes involved in dog coats and figured out how they work together, rather than the genes themselves,” said Elaine A. Ostrander, Ph.D., chief of the Cancer Genetics Branch in NHGRI’s Division of Intramural Research. “We think this approach will help pinpoint multiple genes involved in complex human conditions, such as cancer, heart disease, diabetes and obesity.”

Artificial selection, at the heart of breeding for desirable traits in domesticated animals, has yielded rapid change in a short span of canine history. While researchers estimate that modern dog breeds diverged from wolves some 15,000 years ago, the genetic changes in the dog genome that create multiple coat types are more likely to have been pursued by breeders in just the past 200 years. In fact, short-haired breeds, such as the beagle, display the original, more wolf-like versions of the three genes identified in the study.

Modern dog breeds are part of a unique population structure, having been selectively bred for many years. Based on this structure, the researchers were able to break down a complex phenotype — coat — into possible genetic variations. “When we put these genetic variants back together in different combinations, we found that we could create most of the coat varieties seen in what is among the most diverse species in the world — the dog,” Dr. Ostrander said. “If we can decipher the genetic basis for a complex trait such as the dog’s coat, we believe that we can do it as well with complex diseases.”

–Excerpt from a National Institutes of Health (NIH) News Release published August 27, 2009

Answer the following questions to check your understanding of the reading.

1. How many genes account for the wide variety of coat types in dogs?

2. In two or three sentences, describe how scientists identified these genes.

3. Why do you think it is important to analyze the DNA of many dogs when doing this research?

4. Humans have SNPs too. In general, how might GWAS studies with dogs benefit humans?

Let’s explore how a GWAS works using a simple example that compares two groups of dogs: dogs with black fur and dogs with white fur. Table 1 shows the dogs’ SNP alleles at 17 specific locations in the genome. These specific locations in the genome are called loci (singular: locus). The SNP alleles at each locus are represented by two nucleotides, one from each parental chromosome.

Table 1. SNP alleles at 17 different loci in dogs with black fur (first four rows) and dogs with white fur (last four rows).

If a SNP is found much more frequently in dogs with white fur than in dogs with black fur, the SNP is associated with the white fur color.

1. Give two possible reasons for why a SNP would be associated with a trait like fur color.

To determine whether any of the SNPs in Table 1 are associated with fur color, you can compare the SNPs of the dogs with black fur to those of the dogs with white fur. A SNP is completely associated with fur color if all dogs with white fur share the same alleles at that position, and all dogs with black fur share different alleles at that position. A SNP that is completely associated with a trait is likely located within or close to a gene responsible for that trait.

1. Which SNP in Table 1 do you think is completely associated with fur color? Explain the reasoning for your choice.

A SNP is completely unassociated with fur color if its alleles occur with equal frequency in dogs with black fur and dogs with white fur. A SNP that is completely unassociated with a trait is unlikely to be located within or near the gene responsible for that trait.

1. Which SNPs in Table 1 do you think are completely unassociated with fur color? Explain the reasoning for your choices. (Hint: There are five in total.)

The other SNPs in Table 1 have varying strengths of association with fur color. You’ll learn more about how to evaluate the strength of an association in the next part of this activity. For the question below, make your best guess based on what you’ve learned so far.

1. Which SNP in Table 1 do you think has the next strongest association with fur color, after the completely associated SNP you identified in Question 10? Explain the reasoning for your choice.

Now you know the basic idea of how to identify SNPs associated with certain traits. Scientists look in the region of the associated SNPs to find genes that may be responsible for the traits. So which genes are responsible for the dog coat traits that you explored?

Using GWAS, scientists analyzed the DNA from over 1,000 dogs from 80 recognized breeds with different coat types — including long and short coats, curly and wire (stiff, rough hair) coats, and coats with furnishings (tufts of hair over the eyes and around the mouth). Their study also included several gray wolves, which are ancestors to modern domesticated dogs. Gray wolves have short, straight coats without furnishings.

The scientists identified the three SNPs that had the strongest associations with different coat phenotypes. These SNPs occur within the genes FGF5, RSPO2, and KRT71, which are the three genes that you read about in the news release at the end of Part 1. Each of these three genes has multiple alleles. They each have an ancestral allele, which is the version of the gene found in gray wolves. They also have more recent alleles, which are versions of the genes found in some dog breeds. The more recent alleles differ from the ancestral alleles by single-nucleotide changes in their DNA.

Figure 5 below shows how various combinations of ancestral and more recent alleles account for the seven major coat phenotypes of purebred dogs.

1. Which dog breed in Figure 5 has the ancestral allele for all three genes, similar to gray wolves?

2. Which dog breeds in Figure 5 have a more recent allele for FGF5 but an ancestral allele for KRT71?

You can use Figure 5 to figure out which alleles are associated with certain coat types. For example, if you look at the alleles of the FGF5 gene in different dog breeds, you can see that the ancestral allele (–) is associated with short coats, and the more recent allele (+) is associated with long coats.

1. Which coat type is the ancestral allele of the KRT71 gene associated with?

Identifying which genes affect dog coat types is important for dog breeders, but there are benefits beyond dog breeding. It turns out these genes produce proteins that regulate a variety of processes in all mammals, not just coat variations in dogs. For example, the FGF5 gene (full name fibroblast growth factor 5) plays a role in embryonic development, cell growth, morphogenesis, tissue repair, and tumor growth.

1. How might understanding the functions of genes in dogs help us better understand human health?

2. The methods described in this activity can be used to study the genes of many different organisms. Pick an organism other than dogs or humans that you are interested in. Describe a specific problem or question that you could investigate by doing a GWAS with the organism you picked.