This lecture deals with ancient DNA and archaeological research concerning DNA, it is not my field of expertise and it is quite possible that if something seems amiss or odd it is due to fact that I have misunderstood it, if so please send me a mail or make a comment so that I can correct it.
The fourth lecture of the day was held by Anna Linderholm, PhD student at the Archaeological Research Laboratory (AFL), Stockholm University.
“Ancient DNA and Pathology”
Anna started the lecture with a question; what is DNA?
There are different types of DNA, Chromosomal DNA or nuclear DNA (nDNA) and Mitochondrial DNA or mtDNA. nDNA is the most common DNA used in forensic examinations and mtDNA is most often used while dealing with Ancient DNA. mtDNA is located in organelles called mitochondria while most other DNA is found in the cell nucleus.
One way to find diseases through DNA is to identify DNA from Prokaryote Bacteria. They are a group of organisms that lack a cell nucleus, or any other membrane-bound organelles. Most are unicellular, but some prokaryotes are multicellular organisms. Their DNA can range from 12-160’000 base pairs (bp) (The size of an individual gene is often measured in bp as DNA is usually double-stranded). Bacterias genetic material is typically a single circular chromosome located in the cytoplasm in an irregularly shaped body called the nucleoid.
The extraction process
An ancient DNA laboratory is a sterile environment, it is important that the DNA does not get contaminated. DNA can be extracted from bone and teeth but also coprolites or soft tissue as hair from a mammoth.
When extracting DNA from bone or teeth one normally starts with using a drill and then one separates the organic material from the inorganic material. After this one has to purify the material and multiply it.
To multiply DNA a method called Polymerase Chain Reaction (PCR) is used, basically this means that you create an artificial DNA cell.
Step one is to dye the DNA, when this is done the analyst for the first is able to see if he or she has extracted any DNA. Though if there is DNA he or she can say if it is the right kind of DNA.
What diseases have been identfied through analysis of ancient DNA? Tubercolosis is the most studied so far others are leprocy, palgue, kolera and syphilis etc. These are all tracable through pathogenic bacterias (e.g. bacterias that can cause disease).
There are several examples of projects that have given intresting results. At AFL several intersting project has been carried out, for example:
“Kronan” – Tuberculosis
Emilia Nuorala worked on samples from the man-of-war Kronan. Kronan sunk in the Baltic Sea outside of Öland in battle with a Danish Dutch fleet during the Scandia war in 1676. Ca 800 died and 42 survived the battle. From the excavation some 400 kg of bone as been rescued, these bones are in excellent condition for DNA analysis as they have been lying in darkness and in an oxygen free environment.
20 samples have been tested for TB using PCR, in 9 samples from 3 individuals DNA was successfully extracted. The analysis showed that it was possible to find ancient Mycobacterium tuberculosis in skeletal material that showed no morphological indications of tuberculosis, as in the Kronan case.
Björned – Leprosy and TB
Emilia Nuorala also did an analysis on skeletal remains excavated at Björned, an early Christian cemetery dated to 1000-1100 AD.
Five individuals were tested, one had leprosy and two had TB, one of these individuals had both diseases.
Yersinia Pestis (Pasteurella pestis) – The Palgue
Y. Pestis is caused by bacteria and therefore it should be detectable through DNA analysis. There are three pests
– Antiqua Pestis
– Mediaevalis Pestis
– Orientalis Pestis
The Plague of Justinian was a pandemic that afflicted the Byzantine Empire in 541-542 AD. Samples from two individuals were tested for plague. Both died in 542 AD according to the tombstones. The test was made on teeth (M3 + M1) and on ribs. The test was not positive for plague – why?
- – These two individuals were not affected by plague
- – Due to taphonomic processes the DNA has not survived
- – The extraction didn’t work
Protozoa – Malaria
Protozoa Plasmodium falciparum is a parasite that causes malaria in humans. They multiply in the liver and then in the red blood cells.
Interstingly evidence Malaria has been found in Uleborg, Finland, from the 17th century. There are written sources that describes people who died of fever, that might have been caused by malaria. It is known that malaria was usuall in that area until the end of the 17th century. Anna Linderholm has analyised 30 samples but the analysis are not finshed yet.
Other possiblities in the future
Through DNA analysis we should be able to identify viruses for example CMW, Herpes and Smallpox etc. The oldest records for smallpox is written sources dating to ca 600 AD but the disease could be as old as 10000 BC.
Another disease that is interesting is syphilis. It is hard to find ancient DNA of syphilis as it preserves badly. One of the oldest finds in Europe is that the remains of a medieval woman found in Essex, England, dating to circa 1296-1445. Interesting research is being made on materials from the Easter Island; syphilis came there with Europeans in the 18th century.
The limitiations for research of ancient DNA is preservation and contamination. Ancient DNA is fragmented or degrated and as it degrates it alters, the finds are tahta of small amounts and there is always a risk that the DNA is contaminated.
A case study
This project was started by Ander Götheström and has been finished by Anna Linderholm. It is an indirect study of disease, as it concerns a human gene called CCR5Δ32. One aim has been to find if the gene is in the Scandinavian (Swedish) gene pool during the Neolithic another to establish its geographical spread.
Individuals homozygous for CCR5Δ32 are protected against HIV infection. The geographical distribution of the gene is also interesting. More or less it is only found in European populations and has an especially high frequency in Scandinavia and Sweden.
The gene CCR5Δ32 is a mutation, the mutation or deletion has been dated to somewhere between 700 yrs ago to 3500 yrs ago. The gene has been identified in individuals found in a medieval plague mass burial. This find gives an indirect dating; the deletion is older than 700 yrs.
The gene is only present in Europe (in any measurable quantities) where 16-3 % of the population has it. The highest frequency is found in parts of Norway, Sweden and the Baltic states.
This study has analyzed bone material from the Mesolithic and Neolithic. In total 46 individuals have been analyzed. The Mesolithic finds came from Skateholm and Huseby-Klev and the Neolithic finds are from the Funnel Beaker Culture (TRB) ca 4200-2800 BC and the Pitted ware culture (GRK) ca 3200-2300 BC. Among the others the finds are from Visby, Jettböle, Dragby, Ire, Hjelmars rör and Rössberga.
In this study, two methods has been used to extract DNA. Ander Götheström used the guanidium method and thiocyanate extraction while Anna Linderholm used a method called fishing extraction to find the nuclear marker CCR5Δ32.
In 19 of the 46 individuals they successfully extracted DNA and in 10 of these they identified the CCR5Δ32 gene. These individuals came from at least two Neolithic sites and from Skateholm (As I understood it the dating of the individual is not entirely certain), which is from the Mesolithic.
One conclusion is that approximately 17 % of the Neolithic individuals hade the gene, compared to today’s 14 %.
Why is the frequency so high?
The bottleneck theory is one possibility. For example an epidemic happens and those with the CCR5Δ32 gene survives as do a part of those who don’t have it. But the bottleneck increases the percentage of the population with the CCR5Δ32 gene.
Anna Linderholm holds the smallpox theory as the most likely but there are also others for example the plague theory and the Viking theory.
The Palgue theory does not hold as the CCR5Δ32 does not protect against Yersinia infection, and so it would not have conferred a survival advantage.
The Viking theory is based upon the fact that it is most usual in Scandinavia and that the mutation can be found in the areas the Vikings traveled.
The smallpox theory is that the mutation increased in prevalence as the result of smallpox epidemics. It has been suggested that it may have provided the selective pressure for the CCR5Δ32 mutation.
The findings in this study prove that the mutation is more than 4000 yrs old and possibly older than 6000 yrs.
The End of this lecture, lhe last will come soon (Caroline Arcini)
Remember that these are notes that will be edited, I urge you to comment on anything that seems to be out of place or faulty so that I can correct this. The intention is to compress these notes into a far shorter and more focused version to be printed in the Swedish Osteological Associations journal Benbiten during 2008.