Ancient DNA (aDNA) analysis

Ancient DNA (aDNA) analysis is a new research tool with many applications in fields ranging from genetics through emerging diseases to forensic medicine. aDNA techniques provide a unique opportunity to retrieve genetic information about past populations unavailable by any other approach.
The long-term goal of the present research is to establish a "stratigraphy of genetic profiles" of the diverse populations which inhabited Israel in the past, untangling their inter-group relationships as well as their genetic links to other cultures in the Middle East and Europe.

Projects:
a) population origins and movements in the Southern Levant;
b) origin and spread of genetic disorders in past populations of the Mediterranean Basin;

Infectious diseases in the past
In ancient Europe, tuberculosis was one of the most widely prevalent infectious diseases. Incidence of bone pathology in skeletal remains from medieval Lithuania suggests that 18-25% of the population suffered from the disease. We have detected the presence of Mycobacterium tuberculosis in skeletal remains from Lithuania, dated to the 15th to 17th centuries by amplifying a part of a repetitive insertion element-like sequence (IS 6110). DNA of the bacillus has been identified both in pathological and normal tissues (bones and even teeth) of the same individuals, proving hematogenous spread of bacilli. Moreover, presence of M. tuberculosis DNA has been demonstrated also in skeletal remains of individuals without specific lesions. The results indicate that a much higher percentage of individuals were infected than previously thought. Our findings open the possibility of examining the actual prevalence of tuberculosis in ancient populations from collections in which individuals are represented by single bones or teeth.

Forensic and archaeological applications
Gender determination of skeletal remains has considerable importance on forensic and archaeological studies. However, the accuracy of identification from morphometric analyses is limited in the case of fragmentary remains of adults, or even complete remains of children. Blind studies carried out on specimens of known gender, have demonstrated that while correct identification may reach as high as 90-95% for complete adult skeletons, it may fall to 60% or less when dealing with fragmentary remains or infant skeletons. New developments in molecular biology have provided alternative and reliable methods for gender determination based on identification of DNA sequences specific to the X and/or Y chromosomes.

Gender and Burial customs
Today archaeologists are paying increasing attention to examining social structure within past societies. Archaeological studies of gender differences especially in regard to children have been traditionally explored through identification of grave goods considered indicative of female or male roles. Physical anthropology expands the study of mortuary practices through direct identification of sex, even when grave goods are absent. The reliability of such analyses varies with the condition of the bones. For a complete adult skeleton it approximates 90-95%. However, in fragmentary remains, or those of infants, the reliability of sex identification falls as low as 60%.

Recent developments in molecular biology in analyzing DNA recovered from ancient bones have provided reliable methods for gender determination based on amplification of DNA sequences specific to the X and/or Y chromosome (Faerman et al., 1995). Population structure, male and female status in past societies, and gender differences in burial practices can now be attempted even on fragmentary skeletal remains as well as those of infants and children. In Israel, these infant remains may have been treated with great care, as for example, the jar burials with grave goods found at Tel-Teo (Kahila Bar-Gal & Smith 2001), or alternately, with complete disregard, like the infants thrown into sewers in Late Roman Ashkelon (Smith & Kahila, 1992).

Out of the 17 burials found at Tel Teo, the Huleh valley, 10 were infants, with 6 of them dated to the pottery Neolithic and the remainder dated to the Chalcholithic and Early Bronze Age. Nine of these were subjected to DNA-based sex identification (Smith et al, 1999). The results were consistent with all five specimens (of nine), which yielded amplifiable DNA, being male.

This method was applied to clarify the social basis of infanticide in Late Roman - Early Byzantine Periods (Faerman et al, 1997; 1998). Skeletal remains of some 100 neonates were discovered in a sewer, beneath a Roman bathhouse, which might have also served as a brothel. Written sources indicate that in ancient Roman society infanticide was commonly practiced, and that females were preferentially discarded. DNA-based sex identification of the 43 infant left femurs provided results in 19 specimens (14 males and 5 females), indicating that both male and female infants were victims of infanticide in Ashkelon. These findings suggested that the infants might have been offspring of courtesans, serving in the bathhouse, supporting its use as a brothel. Later this research was expanded to Roman Britain (Mays & Faerman 2001).

Genetic history of modern Israeli populations
We examined the genetic relationship among three Jewish communities, the Ashkenazi, Sephardic and Kurdish Jews, who were geographically separated from each other for many centuries. By comparison with Y chromosome haplotypes of other Middle Eastern populations we asked how the Y chromosomes of Jews fit into the genetic landscape of the region. A sample of 526 Y chromosomes representing six Middle Eastern populations (Ashkenazi, Sephardic and Kurdish Jews from Israel, Moslem Kurds, Moslem Arabs from Israel and the Palestinian Authority Area, and Bedouins from the Negev) was analyzed for 13 binary polymorphisms and six microsatellite loci. The investigation of the genetic relationship among three Jewish communities revealed that Kurdish and Sephardic Jews were indistinguishable from each other, while both differed slightly, yet significantly, from Ashkenazi Jews. The differences in Ashkenazim may reflect divergence due to genetic drift during isolation and/or low-level gene flow from European populations. Admixture between Kurdish Jews and their former Moslem host population in Kurdistan appeared to be negligible. In comparison with data available from other relevant populations in the region, Jews were found to be more closely related to groups from the north of the Fertile Crescent (Kurds, Turks and Armenians) than to their Arab neighbors. The two haplogroups Eu 9 and Eu 10 constitute a major part of the Middle Eastern Y chromosome pool. Our data suggest that Eu 9 originated in Turkey and Eu 10 in the southern part of the Fertile Crescent. Genetic dating yielded estimates for the expansion of these haplogroups that mark the Neolithic/Chalcolithic Period in the region. Palestinian Arabs and Bedouins differed from the other Middle Eastern populations studied here mainly in specific high-frequency Eu 10 haplotypes not found in non-Arab groups. We postulate that these chromosomes were introduced through migrations from the Arabian Peninsula over the last two millennia. This study contributes to the elucidation of the complex demographic history that shaped the present-day genetic landscape in the region.
 

Nebel, A., Filon, D., Weiss, D., Weale, M., Faerman, M., Oppenheim, A., Thomas, MG. 2000. High-resolution Y chromosome haplotypes of Israeli and Palestinian Arabs reveal geographic substructure and substantial overlap with haplotypes of Jews. Human Genetics 107: 630-641.
Nebel, A., Filon, D., Hohoff, C., Faerman, M., Brinkmann, B., Oppenheim, A. 2001. Haplogroup-specific deviation from the stepwise mutation model at the microsatellite loci DYS388 and DYS392. European Journal of Human Genetics 9:22-26.
Nebel, A., Filon, D., Brinkman, B., Majumder, P., Faerman, M., Oppenheim, A. 2001. The Y chromosome pool of Jews as part of the genetic landscape of the Middle East. American Journal of Human Genetics 69(5):1095-112.
Nebel A, Landau-Tasseron E, Filon D, Oppenheim A, Faerman M. 2002. Genetic evidence for the expansion of Arabian tribes into the Southern Levant and North
Africa. American Journal of Human Genetics 70(6):1594-6.
Nebel, A., Filon, D., Faerman, M., Soodyall, H., Oppenhei, A. 2004. Y chromosome evidence for a founder effect in Ashkenazi Jews. European Journal of Human Genetics (in press).

Health and Disease Studies in Past Populations
Investigations of human skeletal remains allow reconstruction of nutritional status and general health from birth to death. Physical anthropologists have developed many techniques for assessing health conditions and for analyzing their possible causes. Methods used include measures of health during childhood, such as stature, location and severity of dental enamel defects, cortical bone thickness and the presence or absence of growth arrest lines. Differential diagnosis of trauma or disease affecting the skeleton include morphological, roentgenological and histological techniques, chemical and ancient DNA analyses.

Hand amputation. A complete skeleton of an adult male, aged 45 years was discovered in one of the MB-II shaft tombs excavated in the village located in the Refaim valley. The right hand was missing and the right radius and ulna foreshortened and fused distally.

Amputation of the hand. The distal ends of the radius and ulna are fused in this 45- year-old male found in a shaft tomb in Jerusalem dated to the Middle Bronze I Period (~3800 B.P.), presumably following amputation of the hand. Photograph shows the right radius and ulna with shortening and rounded fusion of the distal ends (Bloom et al. 1995).

No evidence of significant periosteal reaction, cloacae or distruction was observed. Radiography demonstrated firm bony union of the distal shafts of the right radius and ulna.

Radiograph showing firm bony union of the distal ends and absence of infection.

The amputation occurred in adult life as evidenced by the full development and normal cortical thickness. The appearances are thus consistent with good healing, following surgical amputation of a healthy hand or one traumatically damaged, and is likely to have occurred at least 1 year prior to death. This case is the earliest example of limb amputation originating from Israel (Bloom et al. 1995).

Skull trephination. During the excavations at Arad a tomb cave dated to the Early Bronze age period was discovered which contained several secondary burials. The cranial bones of one the individuals, a young male aged 16-18 years, showed scars from an old trephination. A large shallow symmetric depression with shallow sloping edges was observed on both parietal bones.

Trephination: Arad, upper view of the cranial vault. Two symmetric depressions, resulted from scraping with a sharp object are seen on the parietal bones of the skull of a 16-18 male individual from the Early Bronze Age II period (~4000 B.P.) (Smith 1990).

The size and form of the lesion suggest that this was a trephination carried out by scraping. The well-healed lesions show that the death was probably unrelated to the operation and occurred at least one to two years post-operation (Smith 1990).

The Goliath Injury. A healed depressed fracture was found in a Samaritan male aged about 35 years and dating from the 4th century B.C. This fracture was located in the center of the forehead and had a smooth hemispheric shape.

Frontal view of a cranial vault of a Samaritan male (2300 B.P.) aged +35 years showing a healed depressed fracture in the center of the forehead. It was possibly caused by a round, smooth missile delivered with considerable force (Bloom, Smith 1992).

A lateral radiograph of this depression demonstrated it to be a well-healed depressed fracture of the frontal bone with no signs of associated osteomyelitis.

Roentgenogram of the fracture.

The smooth concave appearance of the depression suggests its causation by a round smooth missile delivered with considerable force. This injury may have been the result of a slingshot wound similar to that of Goliath who was felled by a stone from the sling of the young David (Bloom and Smith 1992).

Ancient DNA analysis is a new research approach with many applications in fields ranging from genetics through emerging diseases to forensic medicine. Our laboratory has pioneered this field in Israel. The research has been mainly focused on the genetics of past and present populations of Israel, Middle East and Europe with the main emphasis on population origins, disease patterns and host-pathogen relationships.
Anemia. The potential and reliability of DNA analysis for the identification of human remains were demonstrated by the study of a recent bone sample, which represented a documented case of sickle cell anemia (Faerman et al. 2000). β-globin gene sequences obtained from the specimen revealed homozygosity for the sickle cell mutation, proving the authenticity of the retrieved DNA. Further investigation of mitochondrial and Y chromosome DNA polymorphic markers indicated that this sample came from a male of maternal West African (possibly Yoruban) and paternal Bantu lineages. The medical record, which became available after the DNA analyses had been completed, revealed that it belonged to a Jamaican black male. These findings are consistent with this individual being a descendent of Africans brought to Jamaica during the trans-Atlantic slave trade.

It is commonly assumed that the endemic disease load increased with the establishment of large sedentary settlements, but there is little direct evidence of the pathogens involved. Several hereditary diseases, primarily hemoglobinopathies, are highly prevalent in populations residing in regions that have been infested by malaria. β-thalassemia is widespread in sub-tropical malarial regions, the Mediterranean basin and the Middle East. It has been suggested that the disease provided genetic protection against malaria. Claims for the occurrence of thalassemia in prehistoric populations have been made on the basis of skeletal pathology (porotic hyperostosis). We reported on detecting a known mutation in the β-globin gene in the skeletal remains of a child with severe porotic hyperostosis (Filon et al., 1995). This study is the first and so far the only direct proof that porotic hyperostosis, observed in the skeletal remains found in the Mediterranean region, is a result of genetic anemia.

The shift to agriculture and animal husbandry revolutionized the frequency and number of contacts amongst people and between them and animals. It is generally assumed that, as TB probably occurred as an endemic disease among animals, the first human TB cases may have been contracted from cattle, and Mycobacterium bovis, the agent of cattle TB, was the most likely first infecting organism.
Many clinical forms of tuberculosis leave no specific traces on the human skeleton. Infectious diseases can be traced through identification of DNA of a specific pathogen from human remains. We detected the presence of Mycobacterium tuberculosis in skeletal remains dated back to the 1 millennium A.D. from North-Eastern Europe by amplifying a part of a repetitive insertion element-like sequence (IS 6110) (Faerman et al., 1997). DNA of the bacillus were identified both in pathological and normal tissues (bones and teeth) of the same individuals, and also in skeletal remains of individuals without specific lesions. The results suggested that a higher percentage of the individuals might have been infected than can be estimated from the incidence of bone pathology. These findings open the possibility of examining the actual prevalence of tuberculosis in ancient populations from collections in which individuals are represented by single bones or teeth.