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Will a cemetery excavation establish a link between the Black Death and resistance to AIDS
FROM THE START, Nico Arts sensed that the frail remains of a child buried in front of a medieval church altar had an important story to tell. Arts is the municipal archaeologist in Eindhoven, a prosperous industrial city in the southern Netherlands whose medieval streets vanished long ago beneath a modern warren of concrete and steel. In the late winter of 2002, Arts and his team were conducting a test excavation near St. Catherine’s Church in the city center, in advance of the development of a new public square. As the archaeologists gently troweled away the sediments, they stumbled across graves from the medieval church and its adjacent cemetery. The discovery surprised Arts because human bone rarely survives for centuries in the region’s acidic soils. So he continued digging. At the bottom of the trench lay a pierced silver coin and the skeleton of a sickly, fourteenth-century child who had been laid to rest inside the church walls, an area reserved for the nobility.
Arts believed he had found someone very important, such as the son or daughter of a former lord of Eindhoven. He was certain that the Dutch public would want to know more about this young aristocrat, so he arranged for an artist to reconstruct the child’s face. The artist, however, needed to know the child’s sex — something physical anthropologists could not determine. So Arts persuaded forensic scientists at the University of Louvain in Belgium to test for ancient DNA. The chances of finding it seemed slim because water dissolves DNA, and the repeated pumping of groundwater from underground parking lots over the past decade could have periodically soaked the child’s bones. Bur in 2004, Arts received some unexpected news: one of the child’s milk teeth yielded DNA. It was the first time usable amounts of this molecule had been recovered from an ancient body in the Netherlands, and it showed the child was a boy.
This chance discovery of ancient DNA has led to one of the most ambitious archaeological projects ever to come out of the Netherlands — a massive excavation in the St. Catherine’s Church cemetery and the establishment of a major ancient human DNA databank. With $3.4 million in funding, Arts and a team of archaeologists and physical anthropologists have now unearthed the skeletons of more than 750 Eindhoven citizens. And over the next two years. University of Leiden geneticist Peter de Knijff will attempt to recover DNA from these remains. “We expect that at least 75 percent of all individuals will have ancient DNA and proteins,” says Arts.
For researchers, the Eindhoven DNA bank could prove a major windfall, paving the way for a host of new studies. To unravel the mysteries of human disease, researchers are increasingly studying genetic variations in human populations that increase the risk of illnesses, such as diabetes, or boost resistance to infections such as malaria. By studying the variants over time, researchers hope to advance knowledge of these diseases and gather clues to produce vaccines or new drug treatments. And such medical research is where the Eindhoven DNA bank, which spans 600 years of history, could really shine. “The Dutch initiative seems very important and interesting,” notes Pardis Sabeti, a medical researcher who studies human genetic diversity and disease at the Broad Institute of MIT and Harvard University.
The Dutch team hopes, for example, that their project will reveal the origin and prevalence of a genetic variant that increases resistance to one of the world’s most lethal viruses — HIV. Today, nearly 10 percent of people of northern European descent possess this variant, known as the CCR5 32 allele, and the discovery is sparking the development of a new class of AIDS-fighting drugs. Evidence suggests that this mutation first arose 3,100 to 7,800 years ago, but how did it become so prevalent across Europe in an age before the AIDS epidemic Could this mutation also have boosted resistance to an earlier epidemic, such as smallpox or the Black Death In search of new data, Knijff and his team will search for this variant in the DNA of Eindhoven’s citizens. “There is no doubt that these studies are valuable,” says Susan Scott, a University of Liverpool historian who has written extensively on the Black Death and its possible connection to the HIV-resistance variant. “Whilst I don’t think [ancient DNA] studies will yield a vaccine for AIDS, they may assist molecular geneticists to develop some gene therapy.”
For Arts and many of his colleagues, the bold project has ushered in a new era in archaeological research. “It makes archaeology much more relevant than it ever has been before,” says Eveline Altena, a University of Leiden doctoral student in charge of recovering ancient DNA from the Eindhoven burials. “If we can use archaeological samples to answer medical questions, how cool is that”
ON A SUNNY MORNING IN EARLY JUNE, NiCO Arts hustles down the corridor of the former police station that now houses the Eindhoven Archaeology Centre. In a corner office, physical anthropologist Leonie Korthorst pores over crumbly bones from one of the graves, examining them for telltale traces of disease or injury. Elsewhere in the building, archaeologists are studying detailed maps of the graveyard and analyzing finds ranging from the tiny bone beads of paternosters, an early form of rosary, to a rare medieval copper stylus for writing on wax. And ream members are still screening sediments from the graves.” We had 8,000 kilograms [17,600 pounds] of sand to sieve,” says Arts. “That’s the part we couldn’t do during the excavation, because we had to hurry to allow construction crews to begin work on the new town square.”
At 54, with a thatch of white hair, trendy black glasses, and elegant brown linen shirt, Arts looks more like an architect than a man who makes his living kneeling in the dirt. Born in Eindhoven, he became the town’s first municipal archaeologist in 1989. Since then, he and his colleagues have continuously stayed one step ahead of the bulldozers, gleaning as much data as possible about medieval Eindhoven — a particularly critical task since two great fires destroyed all written records from that period.
Over the past 20 years, however, archaeology has done much to recover this lost history. As Arts points out, Eindhoven was founded in 1225 at the confluence of two small rivers, in a region of poor sandy soils. A market town from the start, it held a population of 300 by the mid-fourteenth century. Most lived in thatch-roofed wooden houses and earned a living from making and selling woolen felt cloth. As in much of feudal Europe, religion was at the center of their lives. With its tall red-brick spire, St. Catherine’s Church dominated the townscape.
Heading out the door of the archaeological center, Arts leads the way to the old church site, a five-minute stroll from his office. Now barricaded behind a high steel fence, the excavation has been filled in and paved over, in preparation for the new town square. But 18 months ago, this was a scene of intense activity as Arts and his crew carefully peeled away the layers of sediments with trowels and spades. Delving down nearly six feet, they discovered the reason for the excellent preservation of the bones. Both the medieval church and its nineteenth-century successor had suffered serious damage in a series of wars and fires: chalky rubble had repeatedly fallen to the ground, where it neutralized the natural acidity of the sediments and protected the skeletons from decay.
For more than six centuries nearly everyone in Eindhoven had been buried in the old churchyard — an estimated 15,000 in all. The excavation cut through just one corner of the cemetery, but it was enough to yield 750 intact graves and some 250 collections of bones reburied during earlier phases of construction. For Arts, the greatest challenge now is to date the skeletons as precisely as possible. Fortunately, the archaeologists have recovered thousands of pottery sherds. His team currently dates many of the sherds by consulting known chronologies for distinctive pottery types. They will also get thermoluminescence dates from brick foundations of the church and radiocarbon dates from the bones. The researchers will also examine the small number of coins they found for further clues. Only with well-dated DNA samples can geneticists track the historic prevalence of key mutations that confer susceptibility or resistance to disease.
IN THE FORENSIC LABORATORY FOR DNA RESEARCH at the University of Leiden Medical Centre, Altena sits in an empty conference room and lays a binder on the table. Tall and willowy, with straight blond hair, she joined the Eindhoven team two-and-a-half years ago to take DNA samples from the dead. Since then, the 31-year-old has begun working on a doctoral degree in the study of ancient DNA at the University of Leiden and assisting Knijff in setting up a laboratory to analyze the Eindhoven samples.
Most museums and universities, she explains, acquired their collections of human skeletons long before archaeologists understood the importance of collecting DNA. As a result, existing collections are often heavily contaminated with modern DNA from the archaeologists and curators who handled them. So Arts and his team adopted precautionary measures to avoid contamination. They placed each archaeologist’s DNA on file for cross-checking. Then, as each skeleton was exposed, Altena followed strict forensic procedures to collect samples. She set up a small plastic tent over the remains to minimize the possibility of contamination from other archaeologists working nearby. Wearing a white body suit and gloves, and using freshly sterilized tools, she extracted as many as four teeth — usually molars — from each of the dead.
“The DNA is in the roots of the teeth, so it is covered on the top by the tooth enamel and on the bottom by the jawbone,” she says. “That means you have protection from contamination and also conservation of the DNA because it degrades more slowly in the teeth.” After pulling each tooth, Altena examined it visually for signs of disease or abnormality. Then she immediately sealed the tooth in a sterilized tube and stored it in a freezer at minus 68 degrees. In all, she collected 2,584 teeth.
To test the DNA preservation in the collection, Altena traveled last November to the Paleogenetics Laboratory at Johannes Gutenberg University in Mainz, Germany. There, with ancient DNA experts Joachim Burger and Wolfgang Haak, she and a Dutch colleague, René Mieremet, tested five representative samples, including teeth from the oldest layers of the cemetery. For the team, it was a moment of truth. Had groundwater dissolved the DNA in some parts of the cemetery Altena’s preliminary tests revealed that all five samples contained both DNA from the cell nucleus and from the mitrochrondia, an organ within the cell that has its own DNA. It was a very promising sign. Such high-quality DNA could provide answers to many complex medical questions — including, perhaps, the genetic history of resistance to AIDS, a disease that killed 2.9 million people worldwide in 2006 alone.
GENETICISTS DISCOVERED THE CCR532 GENE VARIANT in the mid-1990s when researchers observed chat a small percentage of high-risk individuals — including drug users who shared their needles and gay men who had sex with many partners without wearing condoms — did not become infected with HIV. Indeed, this select group seemed to possess a strong natural resistance to the virus. On investigating further, medical researchers in Belgium and the United States discovered that this resistance came from a genetic mutation that prevented HIV from docking onto the cells of the human immune system and infecting them. People who possess one copy of the CR532 mutation have a 70 cent lower risk of HIV infection than people without it. People with two copies are virtually immune to the most prevalent strain of HIV.
Today researchers wonder whether this mutation could also have helped people survive an earlier epidemic, allowing them to pass on the gene to their descendants. One popular theory suggests that CCR532 could have conferred resistance to smallpox or the Black Death.
The Black Death first reached Europe’s shores in A.D. 1347, when Genoese merchant ships pulled into the port of Messina in Sicily. The frightened crews had just fled from the plague-ravaged Crimea; by the time the ships reached Sicily, their crews had black, egglike swellings under their arms and in their groins, and small black blisters stippled their skin. Some raved deliriously, bled from the nose, vomited blood, and exuded a strange, repulsive odor. As most perished, panic spread among Messina’s citizens. Abandoning their houses, they roamed the countryside in a vain attempt to escape infection. “The disease clung to the fugitives and accompanied them everywhere they turned in search of help,” wrote the Franciscan friar Michael Platiensis just 10 years later, in 1357. “Many of the fleeing fell down by the roadside and dragged themselves into the fields and bushes to expire.”
Most researchers now believe that the Black Death was the bubonic plague. Caused by the bacterium Yersinia pestis, it spread from rats to humans, usually by means of an infected flea. But historical records show that the Black Death occurred in areas such as Iceland, where there were no rats. As a result, historian Susan Scott and University of Liverpool zoologist Chris Duncan suggest that Black Death was a viral hemorrhagic fever, akin to Ebola virus. No one, however, debates its lethal effect. In just three years, the Black Death swept across Europe, as far north as the Arctic Circle, claiming the lives of 40 percent of its inhabitants. And over the next three centuries, it struck repeatedly on the continent, filling graveyards whenever it appeared.
COULD THE SMALL BIT OF GENETIC CODE that gives some people of European descent resistance to HIV today also have saved lives from the Black Death centuries ago It is a fascinating theory, one that Arts and Knijff intend to test with the forensically excavated DNA samples from Eindhoven. As Arts points out, the Black Death first arrived in Eindhoven in either 1348 or 1349. And it returned repeatedly in later centuries. Unfortunately, no surviving documents record the effects of the first plague on the town’s citizenry, but records from the late fifteenth century show that Eindhoven’s population plummeted by 40 percent. “We know that there was a crisis,” says Arts, “so it must have been the plague.” Moreover, Arts and his colleagues found signs that some coffins in the church cemetery were washed with lime, a traditional way of burying the contagious,
Knijff and his colleagues plan to search each DNA sample from Eindhoven for the mutation and chart its prevalence over time. If this genetic coding did confer resistance to the Black Death, those carrying it should have lived longer and produced more children than those who didn’t, thereby increasing the frequency of the mutation over rime. But Altena cautions against too much optimism. Searching for the mutation in the Eindhoven samples will be difficult, she explains.” We have a very small selection [of people in the database] and only 10 percent of the population has that mutation.”
Nevertheless, the Eindhoven project clearly demonstrates the key role that archaeologists will soon be playing in a new field of medical research — archaeoepidemiology. In the months to come, for example, Knijff and his colleagues hope to study genetic variants that increase risk to diseases such as diabetes and cardiovascular ailments. By tracking these variants over time, he explains, medical researchers may be able to assess the extent to which diseases are caused by generic risks and by environmental factors such as diet and levels of physical activity. “Archeoepidemiology, once fully mature, will allow us to estimate effects of genetic variants under different environmental conditions,” he says. To encourage this development, Dutch authorities are now planning to make the collection of DNA samples mandatory for all archaeologists who encounter human remains in their digs. Other European countries may soon follow.
For Arts, the dig in a medieval cemetery has forever changed the way he works, as well as how he thinks about the past. As he relaxes at the end of a warm summer day, he marvels at the good fortune that led him to the grave of the boy buried with the silver coin. “If we had found an adult there,” he says with a broad smile, “we wouldn’t have bothered to look for ancient DNA because we would have been able to tell whether it was a man or woman.”
PHOTO (COLOR): Beneath Eindhoven’s modern skin of brick and asphalt lie the bones of its medieval townspeople. Studying their DNA may reveal the origin of the genetic resistance to AIDS.
PHOTO (COLOR): An excavator uses toothpicks to help map a burial before recording the location of each bone and removing them to the lab for cleaning. Roots from trees planted over the site in 1982 extend through many of the burials, creating a challenge for the archaeologists.
PHOTO (COLOR): The remains of a necklace made of bone, amber, and jet still adorns the throat of a 10- to 12-year-old child who died in the 16th century. Analysis of the child’s teeth showed he or she was seriously ill between ages three and six.
PHOTO (COLOR): Eveline Altena prepares to take a DNA sample from a recently excavated molar. The tooth’s pulp cavity seals off DNA from contaminants, making it the best place to look for intact genetic samples.
PHOTO (COLOR): These copper coins were buried in a leather pouch found in the hand of a 10- to 12-year-old child. They allowed archaeologists to date the burial to the 16th century.
PHOTO (COLOR): People who died of infectious diseases such as the Black Death were often buried in wooden coffins coated with lime. In this burial, the wood has rotted away, but the impression of the planks and a thin layer of lime remain.
PHOTO (COLOR): This forensic reconstruction shows the 10-year-old boy, nicknamed Marcus van Eindhoven, whose DNA sample was the first in the Eindhoven DNA bank. He stands on the spot where the boy was buried.