Tandem Repeats and Morphological Variation

All mammals have basically the same set of genes, yet there are obviously some significant differences that distinguish the various species. Researchers currently think that much of mammalian morphological diversity involves cis regulatory regions, or stretches of DNA outside the actual coding region of a gene that are responsible for switching the gene on and off.

However, an important paper by Fondon and Garner (2004) suggests that there is yet another source of variation: tandem repeats. Tandem repeats are short lengths of DNA that are repeated multiple times within a gene, anywhere from a handful of times to more than a hundred. These sequences are also called VNTRs, or variable number tandem repeats, because different individuals within a population may have different numbers of repeats. VNTRs are relatively easy to detect with molecular tools, and researchers know that populations (humans included) may carry a large reservoir of different numbers of repeats. For example, one person might carry three tandem repeats in a particular gene, while another person might bear 15, with no obvious differences between the two individuals that can be traced to that particular gene. So, the question is, what, if anything, does having a different number of tandem repeats do to an organism?

In their 2004 study, researchers John Fondon and Harold Garner set out to answer this question by first looking for populations that exhibited large and obvious morphological differences between individuals, and then looking within these individuals' genomes to see whether those differences could be correlated with the number of tandem repeats present. The duo decided to use domestic dogs as their population; after all, not only are dogs diverse, but dog breeders are notoriously picky about shape and character, and purebred dogs have been under intense selection for specific attributes for many years.

Once a range of morphologies in a particular trait, such as snout shape, has been identified, one can ask whether this range is reflected in the number of repeats in any genes. Thus, Fondon and Garner examined 142 dogs from 92 different breeds, and they looked at 37 different tandem repeats in 17 genes in each dog. The genes selected were those that encoded transcription factors that were at least suspected of playing a role in the formation of specific morphologies during development. Fifteen of the 17 genes turned out to have multiple alleles that varied in the number of copies of repeats they contained.

Figure 1: Slipped-strand mispairing.

Slipped-strand nucleotide mispairing can generate variation in gene expression. Illegitimate base pairing in regions of repetitive DNA during replication, coupled with inadequate DNA mismatch repair systems, can produce deletions or insertions of repeat units. Bulging in the replicated and template strands gives rise to larger and smaller numbers of repeat units, respectively. The figure shows a strand of DNA (blue) being carried through two rounds of replication.

© 2003 Nature Publishing Group Thompson, N. et al. The value of comparison. Nature Reviews Microbiology 1, 11 (2003). All rights reserved.

Figure Detail

The fact that Fondon and Garner found a substantial amount of genetic variation in tandem repeat number is not at all surprising, because tandem repeats are subject to very high mutation rates. This increased probability of mutation (up to 100,000 times greater than the probability of a point mutation) exists because tandem repeats are prone to a kind of error called slipped-strand mispairing. Tandem repeats contain many copies of the same short sequence over and over, so it is easy for the two strands of DNA to get misaligned in this local region—the GTAC sequence on one strand could base-pair with the first CATG in the other strand, or the second, or the third, for example. If the strands are mispaired, then DNA replicating enzymes can err and either clip off some of the repeats or add extra repeats (Figure 1). This represents a special kind of error in that the DNA changes do not occur in random nucleotides, and they produce only different numbers of repeats. Also, note that this lack of fidelity in copying tandem repeats means that such repeats are only found in regions of genes that can tolerate some variability.

Interestingly, slipped-strand mispairing can be foiled by point mutations, even to synonymous codons, within a tandem repeat. Here, a small change in the DNA sequence gives the replication machinery a local difference that can be used to properly align the two strands. Over time, however, a stable tandem repeat will accumulate these small changes and lose its repeated character. On the other hand, a deletion caused by slipped-strand mispairing can remove a point difference, and subsequent mispairing can then expand the sequence, producing a repeat free of imperfections. Thus, one measure of how much selection for variation has occurred within a tandem repeat is its purity. If there are few interruptions in the perfection of the repeat, there has been much deletion and expansion going on within the sequence throughout its history. Conversely, if there are multiple deviations from perfect repetition, then the sequence has not undergone much length variation in the recent past.

Figure 2

The purity of a tandem repeat sequence is therefore a measure of how much selection for new variants has occurred in an organism's lineage. Based on this principle, Fondon and Garner compared the same repeat loci in humans and dogs, and they found that the dog repeats were purer than the human repeats in 29 of 36 cases; in the other seven cases, the dog and human repeats were of equal purity. This finding strongly suggests that the variations in dogs are not just random, neutral changes, but rather, they are the outcome of recent selection at these loci.

Thus, Fondon and Garner determined that there are multiple interesting gene variants in dogs, and these variants have apparently undergone selection. But what effect do the repeats have? Let's consider two specific genes—Runx-2 and Alx-4—as examples.

The good news about these findings is that they demonstrate another mode by which morphological diversity can be added to a population relatively rapidly, as well as another mechanism for fine-tuning evolution. Because tandem repeats are common in the vertebrate genome, these repeats could clearly be a reservoir of variation and a robust and flexible way to add new variations to populations.

There are some limitations to Fondon and Garner's results, though. First, this study focused on an extreme case: purebred dogs that have undergone very strong selection for specific and, in some cases, outright deleterious characteristics. Thus, we simply don't know how important this mode of evolutionary change is under less-artificial conditions. Second, this study and others like it have revealed only correlations, not experimental perturbations. While these correlations are convincing, at some point in the future, it would be helpful to see direct manipulation of the poly-Q/poly-A ratio in the Runx-2 gene of a collie, for instance, to give it the downturned nose of a bull terrier. Finally, it would also be beneficial to obtain additional correlative evidence through developmental studies of the patterns of Runx-2 and Alx-4 gene expression in dog embryos to see exactly how these variations play out.

Figure 5: Large magnitude repeat length mutations can result in gross morphological change.

(A) Alx-4–/– mice exhibit a duplication of their first digit (as indicated by the arrowhead). (B) A radiograph of the rear paw of a Great Pyrenees shows the typical double dewclaw phenotype specified in the breed standard (as indicated by the arrowhead).

A) © 1998 The Company of Biologists. All rights reserved. B) © 2004 National Academy of Sciences Fondon, J. W., & Garner, H. R. Molecular origins of rapid and continuous morphological evolution. Proceedings of the National Academy of Sciences 101, 18058–18063 (2004) doi: 10.1073/pnas.0408118101. All rights reserved.