Fourth Annual DNA Grantees' Workshop
Monday, June 23, 2003
MORNING SESSION
Research Update Briefings: Prototypes and Products
Elaine M. Pagliaro, Moderator
Biography
MS. PAGLIARO: Our first presentation is on an STR (short tandem repeat) forensic typing system in the domestic cat, presented by Dr. Marilyn Menotti-Raymond. Dr. Raymond is a staff scientist at the Laboratory of Genomic Diversity, National Cancer Institute, in Frederick, Maryland.
The goal of her research is to characterize genetic organization in the domestic cat and to develop genomic resources facilitating and establishing Felis catus as a useful animal model. Her research has focused on generation and development of a genetic linkage map of coding loci and STRs in the domestic cat, and she has also been involved with the application of genetic markers in the cat for forensic analysis.
I know in our laboratory there have been many cases and much interest in doing DNA in domestic animals, so I'm sure that we'll all welcome this particular aspect of forensic testing.
An STR Typing System for Forensic Analysis of Domestic Cat Specimens
Marilyn Menotti-Raymond
Biography
DR. MENOTTI-RAYMOND: Good morning and thank you for the opportunity to come here today and to present our research on an STR forensic typing system in the domestic cat. Menotti: Slide 1
Mankind has had a long-time fascination with the cat, a relationship that was formalized as much as 6,000 to 8,000 years ago, based on skeletal remains of a cat that were found in the ancient ruins of Kurakatea in southern Cyprus. Menotti: Slide 2
This island habitat was not native to the wild cat (Felis sylvestris), from which the Felis catus was domesticated, and so it was felt that the cat was brought there as part of the human entourage onto the island. Menotti: Slide 3
Today, approximately 65 to 70 million cats reside in households across the United States, which translates to about 1 cat to every 3 households. Not only do we take record or evidence of our pets wherever we go, but our animals can act as silent witnesses to those who visit us. A study [D'Andrea, F., F. Fridez, and R. Coquoz, "Preliminary experiments on the Transfer of Animal Hair During Simulated Criminal Behavior," Journal of Forensic Science 43 (6) (1998): 1257–1258.] on the transfer and persistence of animal hairs has shown that it's virtually impossible to enter a home where a domestic animal resides without becoming a carrier of some of its hairs. Menotti: Slide 4
So it's not unusual for animal specimens, particularly hairs, to be part of physical evidence associated with crime scenes. But until recently, the genetic individualization of animal specimens was not possible for a variety of reasons, particularly the lack of species-specific single locus probes, databases, and genetic maps. But we're in the age of the genomic revolution and during the past 10 years, species-specific maps—particularly in all mammalian species that are integral to our daily lives—have been developed. Menotti: Slide 5
We now have linkage and radiation-hybrid maps that include coding loci and STR loci, which have generated tremendous forensic potential for the genetic individualization of animals that are a part of our daily lives.
At the National Cancer Institute's Laboratory of Genomic Diversity, we've had a longstanding interest in the cat, beginning with my boss Steve O'Brien's first interest in the cat as an animal model for viral-induced cancers. I'm sure you're wondering why we're working with forensic sciences at the National Cancer Institute. Menotti: Slide 6
These interests expanded to our interest in the cat as a model for infectious and hereditary diseases which plague mankind. Menotti: Slide 7
Particularly in the past 10 years, the Animal Genetics Section of the laboratory has devoted much effort toward developing both genetic linkage and radiation-hybrid maps of the cat, including coding loci and STR loci, so that we can isolate and characterize genes associated with hereditary pathology that's segregated in diseased cat pedigrees. Menotti: Slide 8
As this audience well appreciates, STR loci have broad applications outside linkage mapping. Menotti: Slide 9
It was actually a graduate student in our lab, Melanie Culver, who first used some of our cat STRs in an animal-related case to identify a puma—from the local California puma population—that attacked a jogger. Actually, the puma had already been shot and killed, but it was good to see that the correct one was identified. Melanie actually had a California puma database.
Soon after that, we became involved with cat hairs associated with a human crime scene in a case that many people have heard about, in which composite profiles that were generated with 10 dinucleotide repeat loci from hairs isolated from a jacket found near the crime scene were compared with a composite profile generated from DNA from the subpoenaed cat, Snowball, of the defendant. Snowball, who became quite well known and who I understand is dead, had nothing to do with the case. Menotti: Slide 10 and 11
So these matching composite profiles are part of the evidence that was introduced into court and led to a second degree murder conviction and our continued interest in developing a forensic typing system in the cat—an interest that has been funded by the National Institute of Justice.
So the first thing we have to ask ourselves is what is required in an animal STR DNA typing system, because we're starting from the bottom up. Menotti: Slide 12
We need a means to identify the species origin of samples. Many hair samples, particularly wool hairs or undercover hairs, are very difficult to identify, even on a species level. This is effected by universal mitochondrial DNA primers, which can amplify mitochondrial DNA and be sequenced to identify the species of the sample. So that was sort of taken care of for us.
We needed a means to quantify DNA yield. Is STR analysis even going to be possible, given the amount of DNA that can be isolated from many animal hair specimens? Menotti: Slide 13
So our goals in developing an STR typing system were to develop an assay to quantify DNA yield and to isolate, characterize, and map STR candidates. Largely we had dinucleotide repeats in our repertoire when we started this project. We had some tetras, but not enough, and selected a forensic panel of 10 STR loci and a gender-specific locus, so a sequence-tagged site that would identify whether it was—particularly we targeted the locus on the Y chromosome.
Then we wanted to develop a multiplex amplification protocol that would be tested and validated and genotyped in a sample collection of 37 recognized cat breeds in the United States to generate a genetic database.
So I want to report on our work with these goals. Here is our criteria for STR candidates. Menotti: Slide 14
Again, we started the project by isolating a bunch of tetranucleotide repeats, and in this respect, I want to acknowledge the work of my chief colleague Victor David. He generated a tetranucleotide-enriched library. In the end, we had isolated about 53 promising candidates that were polymorphic in the cat genome. We needed to show that they exhibited Mendelian inheritance, that they were independent linkage groups, that they would exhibit high heterozygosity in cat breeds and cat populations, and that these last four could be affected by judicious primer design. Menotti: Slide 15
Now, this simply shows the Mendelian inheritance testing which was conducted on one of the loci. I believe this one is in the forensic panel. It was generated in a multigeneration pedigree: sire, dam, and the siblings. All of our loci exhibit Mendelian inheritance.
Then we physically had to map them in the cat genome. This work was conducted in our laboratory by Bill Murphy, who constructed a radiation-hybrid map of the cat that included coding loci and STR loci. He simply incorporated these with several hundred other markers.
This is an ideogram of the cat karyotype. The cat has 19 pairs of chromosomes and this shows the physical location of these markers on the cat chromosomes and ultimately the ones that were selected for the forensic panel. Please note that we selected two of the cat's largest chromosomes, A1 and B1, but they're on independent arms and we know, based on cross-reference of these markers that were physically mapped to those in our genetic linkage map, that they are not linked. Then all of the others were on independent cat chromosomes, and we also have one on D4. Menotti: Slide 16
This shows the cat radiation-hybrid map for the Y chromosome. We elected to use a sequence tag site (STS) from the cat SRY gene to identify male cat samples that would be included in our STR forensic typing system. Menotti: Slide 17
This shows the profiles of 11 loci (and the chromosomes they are located on) selected because they offered leeway in case there were some problems in developing a multiplex. Ultimately, we ended up keeping all of them. Menotti: Slide 18
We did not know the kinds of heterozygosities that we would see in cat breeds, so we took DNA from a subset of our growing sample collection of animals, for which we had a fair abundance of DNA (approximately 230 cats representing 28 breeds), and genotyped 22 promising candidates.
This simply shows the average heterozygosity observed across those 28 breeds for the 11 loci we selected for the panel. A number of things came into consideration in selecting the panel: heterozygosity observed; presence of Mendelian inheritance; map location in the cat genome; and the nature of the repeat motif. It also shows the number of alleles that were observed.
Then we entered into collaboration with John Butler, who you all know. John has developed a multiplex amplification protocol for us. This slide was generated by John and it simply shows the allele size distribution for our 11 STRs. We have a four-dye system and a gender-identifying SRY locus. It shows that the allele sizes spread from 100 to 400 base pairs in the initial composite set of 230 cats. Menotti: Slide 19
This shows a composite profile on amplification of the multiplex in 4 nanograms of male cat DNA. Because this is a male cat, we had a very strong signal from the SRY gene and a little bit of bleed-through. Again, we have a four-dye system and a good balance of products. Robustness is still being examined, but John observes that the panel amplified well at under 0.7 nanograms of DNA, even on multiple platforms. Menotti: Slide 20
This shows the amplification profile in 4 nanograms of female cat DNA. Here, a product is absent at the SRY locus. Menotti: Slide 21
Now, we also examined the specificity of the complex in a range of mammalian species. (We have a nearly frozen zoo at our laboratory.) Although we look at many, many species around the world, we took a sample set of North American mammalian species. We observed products in other members of the Felis family, as expected, and also a few stray products from other members of the carnivore orders.
As expected, we saw two loci amplified in the bear, two in the dog, two in the wolf, and one in the otter. I believe all the loci were monomorphic except for the loci in the bear, which were polymorphic. But outside the carnivore order, given our amplification conditions, we found no products, particularly no amplification products, from human DNA. Menotti: Slide 22
Now, we've also had the opportunity to examine mutation rates for these loci. This is actually a very complicated diagram of a multigenerational cat family maintained by the Nestle-Purina Company. They provided the DNA that we are using to construct an extended linkage map of the domestic cat. Menotti: Slide 23
DNA from 256 individuals and the potential of 483 informative meioses allow us to directly examine mutation rates for the loci. This simply shows the detection of mutation for one locus. So here we have the dam, the sire, and then in this offspring we have the detection of a mutation. So here's the paternal allele, so either we have a gain or two repeats or a loss of one in this maternally transmitted allele. Menotti: Slide 24
This shows sex-average mutation rates observed for six of our loci. The limit of our detection is about 0.2 percent, but you'll observe that for four of these loci, we had a mutation rate of less than what we can observe, somewhere less than 0.2 percent. And the sex-average rate for two of them were about 0.2 percent. This is within the sex-average mutation rate observed for many of the CODIS loci. Menotti: Slide 25
Now I'm going to switch gears and tell you about our sample collection and tell you something about the domestic cat breeds from which we are generating the database. As I said before, the cat has been domesticated for perhaps 6,000 to 8,000 years, and cat breeds are a relatively recent addition to the scene. Menotti: Slide 26
What we know about cat breeds is fairly anecdotal in nature. We know that as many as perhaps 100 cat breeds have arisen at one point and many of these have disappeared. What I want to show you is what we know, at least anecdotally, about when and where the breeds in the database came from.
So these are breeds that have arisen: the older breeds, the breeds that have arisen pre-1800, during the 1800s, and during the 1900s. You'll note that the majority of cat breeds recognized in the United States today have arisen during the past 100 years and many even within the past 40 years. Cat breeds, then, are a very recent addition to the scene. Menotti: Slide 27
Breeds have been selected as the result of artificial selection approximately between 10 and 30 loci. Only one has been characterized on the molecular genetic level for traits of phenotypic distinctiveness that are prized by cat breeders: coat patterns (as with the Bengal ) and coat hair length. Menotti: Slide 28
This is the most popular breed, the Persian, that comprises approximately 60 percent of cat breeds. This is the Cornish Rex, who, minus the guard hairs, has sort of a rough appearance. Menotti: Slide 29
Then we have other qualities of phenotypic distinctiveness. Here we have a recent breed called the Scottish fold. It has folded ears and has only been around, or at least recognized, for about 40 years. This is an older breed, the Japanese bobtail. It has a bobbed tail. This is the Cat Munchkin—the cat version of a Dachshund. I've never even seen one of these cats. There aren't very many of them, so they're not in our database. They're actually a good model for achondroplasia, something ultimately we might be interested in investigating. Menotti: Slide 30
As I said, we were interested in 37 breeds, but I think that we have 38 breeds. We hope to obtain up to 50 individuals in each breed. Some of these have just proved impossible to do so because they are too rare or it involves the cooperation of the breeders. Menotti: Slide 31
Actually, we got tremendous support from the cat breeders. We wanted blood and buccal swab samples and eight-generation pedigrees to determine the relatedness of members in our sample collection. This shows me at a cat show with a cooperative cat owner and a cooperative cat. Shows turned out to be the most productive way of finding some of the rarer breeds. Menotti: Slide 32
We have 1,203 cats among 38 breeds, and this simply shows the numbers for each breed. We have a low in the American Wire Hair, a very recent cat breed with crinkly hair, and more than 50 for some breeds. Menotti: Slide 33
This slide shows the informativeness of our forensic panel of 11 loci across our sample set of 1,200 cats. Genotyping is nearly completed. We've run through it twice to make sure that we're confident about all the application allele calls. There are a few that we want to rerun.
The heterozygosities reported here are averages for the 11 loci across the 38 breeds. They range from a low of 0.56 (for locus D06) to a high of 0.78 (for loci F85 and C08). The number of alleles observed across the entire composite set of 1,200 cats includes a high of 45 alleles and a high range of 124 base pairs for F85—our most informative locus. You note here this is the range of heterozygosities observed across the range for each locus. So for instance, this one particular locus is fixed in the Oriental Shorthair. They only exhibit one size allele, so there is quite a range of heterozygosities. Menotti: Slide 34
With an overall average of 0.71 for the 11 loci, cat breeds are moderately inbred relative to mixed breed domestic cats—actually, I was expecting it to be lower than that. The small sample size of mixed breed domestic cats gave us a value of 0.86. Ultimately a database is going to have to be generated with mixed breed domestic cats.
This shows the range of average heterozygosities across the 38 breeds for the 11 loci. It ranges from a low of 0.4 in the Singapura to a high of 0.77 in the Turkish Angora, Norwegian Forest Cat, and Selkirk Rex. So again, as expected, the average range of heterozygosities is large. Menotti: Slide 35
Even though moderately inbred, the panel shows excellent promise in terms of generating genetic distinctiveness among cat breeds. This slide summarizes the match probabilities. This was done with the smaller dataset. We wanted to be sure of all of the allele calls in the larger database before we generated the match probabilities. Probabilities range anywhere from 10–7 to 10–13 in the Manx. There's one higher (10–14 in the Norwegian Forest Cat), and I believe we saw something like a value in the 10–13 range in the mixed breed domestic cats. Menotti: Slide 36 and 37
This slide describes the relationships of bred cats to one another. It's a phylogenetic tree—a neighbor-joining tree based on 217 cats. Each one of the lines represents an individual. It's generated based on the proportion of alleles shared on a pairwise comparison between individuals. Menotti: Slide 38
It gives you a way of saying, well, what is the relationship of these different breeds to one another and what is the level of genetic distinctiveness. This is sort of what I expected to see based on the level of how long breeds have been around. Breeds of cats with Southeast Asian ancestry (e.g., the Burmese and the Bombay, the Havana Brown, Birman, Oriental Shorthair, California Shorthair, and Siamese) can show genetic relatedness in ways that other breeds cannot.
Outside of monophyletic relationships we observed for the Egyptian Mau and the Abyssinian, the genetic distinctiveness of breeds tends to collapse. There's not a lot of genetic signal there. This is not surprising because, as I said, more than half the breeds have been around for less than 100 years.
This tree was generated with 22 STR loci in the smaller composite dataset, and we're going to be generating a tree with the larger set of 1,200 cats, but we can only do that with the 11 loci in the core panel. We simply do not have enough DNA for many of these animals. Many of the cats from which we got buccal swabs were not particularly cooperative, so we will not be able to generate that tree with 22 loci. So with the 11 loci, given the sample, would you be able to say that's a Russian Blue, that's a mixed breed domestic cat, that's a Ragdoll? No, not with any level of confidence.
The last thing that we've been working on is a means to quantify domestic cat nuclear DNA. Having enough DNA is always going to be an issue when you're dealing with animal specimens. We found that the most we can expect from a cat hair root is about 30 nanograms of DNA. The issue is always going to be, do I have any nuclear DNA here or how much do I have. The issue is not new, but we're starting out with so much less than you start out with, for example, a human hair. Menotti: Slide 39
We've developed a real-time PCR (polymerase chain reaction) detection method in which we make a comparison of the product profile. We generate a PCR product from a scant proportion of the extract from the hair, and then we compare that product profile to that of a dilution standard of reference cat DNA—10 nanograms to a femtogram—to estimate the yield of the total product and the likelihood of success of STR amplification.
We used small interspersed nuclear elements (SINEs) for our targets. We estimated these at 2 million per haploid genome. Just let me show you that we see a linear relationship between DNA concentration down to 10 femtograms with threshold of detection cycle. Menotti: Slide 40 and 41
Right now, we're in the process of validating our quantitative SINE assay, validating our cat multiplex, and generating allelic ladders. I won't read all of these conclusions because my time is up, but I will be happy to answer any questions. Menotti: Slide 42 and 43
I want to thank all of these people in my laboratory. In particular, I want to acknowledge the work of Victor David (my chief colleague and compatriot in this effort) Steve O'Brien (my boss), and John Butler, who has been essential in the development of the STR multiplex. Thank you. Menotti: Slide 44 and 45