Fourth Annual DNA Grantees' Workshop

Tuesday, June 24, 2003

MORNING SESSION

Increasing the Predictability and Success Rate of Skeletal-Evidence Typing: Using Physical Characteristics of Bone as a Metric for DNA Quality and Quantity
David Foran
Biography

MR. TAMBASCO: Our next speaker, Dr. David Foran, earned his Ph.D. in molecular genetics from the University of Michigan. He's currently on staff in the forensic biology program as an assistant professor at Michigan State University's School of Community Justice.

He is going to speak to us today about increasing the predictability and success rate of skeletal-evidence typing using physical characteristics of bone as a metric for DNA quality and quantity.

DR. FORAN: This is a talk on bone and skeletal material. My collaborators on this are Doug Ubelaker and George Washington University. Foran: Slides 1 and 2

The questions we're asking in our work are pretty basic: Is there a correlation between the appearance of an aged bone or skeletal material? How weathered is it? What DNA quantity exists within it? Can there be some predictive value? Foran: Slide 3

Is there a correlation between how this bone appears, what its DNA quality is, and how degraded the material is? That's kind of a general question: Do bone and DNA degrade somehow in a similar fashion? Intuitively, if you were handed two femurs and one was in pretty bad shape and the other one was in pretty good shape, you would go after DNA from the one that's in pretty good shape. It just makes sense. On the other hand, factors that affect how a piece of bone degrades could be very different from how DNA degrades. You could just sit here and think up tons and tons of different ideas about what is causing this degradation process and they may not be different. You can crush a bone, but you're not going to crush DNA.

We're also asking in this study, at any given level of bone weathering and level of degradation or if we had only skeletal material from pieces of bone, whether a particular place on a bone or a particular type of bone is most likely the best source of DNA. Can we have some kind of predictive value? Foran: Slide 4

After that, we have to ask how applicable these are to a wide range of skeletal materials. We have skeletal material from all over the world, different ages, different factors, and so on. If we make up some general rules, will they apply to some of the material that you come across?

Overall, can we use the bone appearance in some kind of predictive manner? You see a piece of bone and say, "Ah, I would predict this based on how it appears." There are many confounders in this kind of study. One is bone age. If you look through the research or the literature, you find studies of 20-, 500-, and 1,000-year-old bones and you say, "Oh, we got DNA from this one, but not from this one." And of course those don't make for the best studies in the world because you have that confounder. Foran: Slide 5

Bone environment is another big one. If you take a piece of bone that's been lying in the desert for 100 years, one that's been lying in the swamp, and one that's been lying in a bog somewhere, you're going to get very different results, obviously, even though they're all the same age. You could think of many things in the environment that could be factored in, for example pH (potential of hydrogen) of the soil and freeze-thaw conditions. And you could think of lots of other things: the ancestry of the person, the age of the person, the sex of the person, and more and more. All of these types of things can complicate this type of work.

So our strategy is to start by trying to eliminate as many of these confounding factors as possible in order to ask the basic question: Is there a correlation between how a bone appears and the DNA within it?

Which takes us to the Voegtly Church, near Pittsburgh, Pennsylvania, and to how this whole study came about. Foran: Slide 6

A lot of Scottish, English, and Irish and a very large number of southern German and Swiss immigrants moved to this area. In 1822, the Voegtly family and several others from Germany together bought about 160 acres of land. They were very religious people, and originally they went across the river in Pittsburgh to the German Evangelical Church. There were some problems and issues there, so in 1833 they founded their own church, First German Protestant Evangelical Church of Allegheny. Eventually it became known as the Voegtly Church because the Voegtlys were the ones who donated the land. It was very much so a German church. Their services were only spoken in German until 1913, and up until 1942 they held part of the services in German.

In 1850, there were famous people on the Allegheny: Andrew Carnegie and Stephen Foster. Then H.J. Heinz moved one of his big processing plants across the street from the church, and it [the church] wasn't quite as popular as it was before that.

When the church was founded, there was a rule that anyone who supported the church had the right to be buried there. People died at a pretty fast rate back then, so a lot of people were buried behind the church in a very short amount of time. Between the founding of the church in 1830 and about 1850, the cemetery filled up.

The church then purchased a piece of land nearby on Troy Hill and moved the cemetery there. So they moved everybody over there and used the Troy Hill location as their church cemetery from then on. The church expanded over time. By 1882, an addition was made to the church, and when automobiles came along, a parking lot was added.

This whole area is now Pittsburgh. Foran: Slide 7

In 1959, the Pennsylvania Department of Transportation (PENNDOT) decided that they wanted to put in an interstate, which is known as the E Street Valley Expressway, to connect the downtown area of Pittsburgh to Interstate Route 79. The Voegtly Church was in the right of way, so the church was condemned and, needless to say, membership declined thereafter.

PENNDOT acquired the property in 1984, and in 1985, the church was leveled. Highway construction proceeded through this area in 1987. When the highway construction was starting, a bit of the church foundation was still there and the parking lot was there. The bulldozers and backhoes came in, started digging, and soon came across five grave sites. Once they hit five, they stopped digging.

It was later figured out through church records what actually happened. Well, let's back up one step. The church records that were available were in this ancient German dialect that nobody spoke. Even people who fluently spoke and read German had no idea what the records said. They didn't know how many people might be there or the locations of graves. There were absolutely no markers. No one had any idea what was going on.

It was later learned that Nicholas Voegtly, Sr., and Elizabeth, his wife, had actually been moved to the Troy Hill Cemetery; everybody else had just been left behind. So the cemetery was treated like a large archaeological dig. The State of Pennsylvania, given the timing of highway construction, brought in a lot of people, and started digging and uncovering graves. In the end, they found more than 700 graves. Foran: Slide 8

Later, church records were translated and showed that about 750 people had been buried there. They didn't find them all. Many of the records had just a person's name or said something like "never saw God" or "never met life" or "never took a breath," indicating a stillbirth or someone who died early on or as a result of a miscarriage. Those bones or graves weren't expected to be found.

The graves were quite close together and more or less randomly distributed. Children were often put in a separate area because they had not yet become evil or whatever, so they could quickly find the path to God. Everybody else had to work a little harder. Foran: Slide 9

People were put in a north-south direction with their hands folded across their stomachs. All of the coffins were simple, six-sided wooden structures made from similar materials. There was a bit of ornamentation but not very much, so that wasn't much of a problem. Foran: Slide 10

Nobody associated with the Voegtly Church at the time it had been condemned knew who the deceased actually were, so there was nobody making claims over the bodies. Because of that, PENNDOT, having 700 bodies and nothing to do with them, asked the Smithsonian Institution if it wanted them, and of course it did.

That's where we came in. We talked to Doug Ubelaker one day at the Smithsonian about various projects. We had several less confounding factors to deal with:

• The bones had all been in the ground about the same amount of time, from a 20-year period (1830 to 1850) up until the day they were excavated.

• The bones were in the same environment, as best as we could tell.

• The ground was pretty flat with similar soil.

• The caskets were right next to each other.

• The samples had gone through the same freeze-thaw cycles.

• The caskets were all the same.

• The sample was the same population of people. Foran: Slide 11

But what they found when they dug up these bones was that the bones had very different levels of degradation. They would find some skeletons that were in really good condition, others right next to them that were highly degraded, and everything in between.

The anthropologists tell me that they have a grading scale for levels of degradation or bone weathering. It's just a scale of zero to five. Zero is the bones that are in very good shape and show no sign of weathering. That's what most of us would have right now, hopefully. Stage 1 is bones that show a little bit of cracking longitudinally. Stage 2 is cracking and some flaking of the bone. Stage 3 is a bone surface with some patches that are starting to wear down into the bone but that still have fibrous material. Stage 4 is a bone surface that is quite coarse and pieces are falling out. You've probably come across that kind of material. Stage 5 is a bone that actually crumbles when you pick it up in your hand. Foran: Slides 12–18

Church records show and Ubelaker and other anthropologists found that a lot of people died at childbirth and at very young ages back then. Here, though, we have a nice distribution of age ranges, both in males and females, available to us. Foran: Slides 19 and 20

All this material comes from Jones and Ubelaker, who have just put out a large monograph from the Smithsonian. It contains details about every single burial, the bones that were found, dentitions, and artifacts—just tons of material on every single burial that was uncovered. Foran: Slides 21–29

With permission from PENNDOT, our students collected samples from the Smithsonian:

• Different bone types from each skeleton, including pieces of long bone, generally femur. We took out a wedge-shaped piece or two wedge-shaped pieces next to each other.

• Teeth when available (they usually were), oftentimes a molar or two.

• Ribs. These were the most prevalent bone, not surprisingly.

• A flat bone, usually a piece of pelvis.

Most of the material, you could pick up and break pretty easily in your hands.

For our study, we're focusing on mitochondrial DNA for obvious reasons, and we're also doing nuclear DNA just to see what we get. We're looking at the quantity of mitochondrial and nuclear DNA in these samples, and we're doing that by weight and by mass of the sample. We're determining the maximum amplifiable piece that we can obtain, so we're going to start with small pieces and amplify progressively bigger pieces in each sample until we can get up to 200, 300, or 400 bases or whatever we can get. Foran: Slide 30

We're then going to look for any statistically different results among these samples based on either the weathering stage or the bone type, for which we have good data for both. Once we see if there is a correlation between the weathering stage, the bone type, and the quantity and quality of DNA, our next step is to determine how applicable this is to other material.

Again, our original goal was to get rid of confounding factors like different ages of bone and different environmental conditions, and in the simplest conditions find out what kind of correlations are applicable to other materials. So if we find a piece of bone that's much older or younger or been in a different environment, we can apply certain rules of thumb for obtaining the best DNA.

We'll obviously take into account other variables that we know about: sex of the individual, its general age, and all other materials that we have. For consistency, one of the controls we're using is the mitochondrial sequencing so that when we have a piece of fragment of DNA from a tooth and a fragment from a long bone we can sequence those and later on check to make sure we're getting the same individual, or at least the same family, but generally the same individual. Foran: Slide 31

We're also doing soil samples. For these, we scrape soil that was readily available from the bones. Forensic chemists are looking at elements like pH, clay content, organic matter, and so on because people felt it was important to look at those things in the general condition or the general environment of the bone.

Ubelaker and I also are collaborating with people in Brazil who are looking at the cellular structure of the material. They're taking sections—and that's why we took two pieces of bone next to each other—and doing electron micrographs of them and looking at the structure of the bone to see how the substructure relates to degradation and how it correlates with our results.

We're also paying attention to differences in grading a whole skeleton. For example, we'd have a stage 2 skeleton, which means that the bones are in pretty good shape, but we'd get a piece of bone (e.g., a rib) that was actually in pretty bad shape. The anthropologist who grades the whole skeleton may be doing stuff very differently from those of us who are working with only tiny little fragments. So we're paying attention to things like that.

These samples were originally donated to the Smithsonian by PENNDOT, as I mentioned, who went to the families and asked if it was okay that the samples were given to the Smithsonian. Everybody was fine with it. We have no idea who they were; we're just glad that they went to science. Then a mortuary in Pennsylvania—who has the rights with the State to take any unclaimed body that was found and cremate it at about $700 a body—found out about this and said, "Hmm, 700 burials at $700 a body," and went back to the families and said, "Shouldn't your loved ones be taken care of in an appropriate manner?" And of course, the families asked for the bones back. Since then, all of the bodies have been returned to PENNDOT, but we still have our samples. Foran: Slide 32

I want to thank all the people in my lab who worked on this—Jen Dreier at George Washington University, who did a lot of the collecting of the samples with help from various undergrads at the Smithsonian—and of course, NIJ for supporting this work. Foran: Slide 33

Thank you very much.