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Florida State University Libraries Electronic Theses, Treatises and Dissertations The Graduate School 2004 The Possible Selection of the Sickle Cell Trait in Early Homo Kellei L. Jefferson Follow this and additional works at the FSU Digital Library. For more information, please contact [email protected] THE FLORIDA STATE UNIVERSITY COLLEGE OF ARTS AND SCIENCES The Possible Selection of the Sickle Cell Trait in Early Homo By Kellei L. Jefferson A Thesis submitted to the Department of Anthropology in partial fulfillment of the requirements for the degree of Master of Science Degree Awarded: Spring Semester, 2004 The members of the Committee approve the thesis of Kellei L. Jefferson, defended on Monday, March 15, 2004. ________________________________________ Dean Falk Professor Directing Thesis _________________________________________ Glenn Doran Committee Member __________________________________________ Elizabeth Peters Committee Member The Office of Graduate Studies has verified and approved the above named committee members. ii ACKNOWLEDGEMENTS I would like to express my appreciation of the faculty members who chose to serve on my thesis committee; Dr. Dean Falk, Dr. Glen Doran and Dr. Elizabeth Peters. Their thoughts and suggestions for the direction of this paper aided me immeasurably. In addition, I would like to thank my incredibly loving fiancé, Seth Johnstone, whose support, encouragement and patience contributed greatly to the completion of this paper. I would also like to acknowledge my mother, who has always been a mentor to me and was instrumental in my choosing to gain this degree. iii TABLE OF CONTENTS LIST OF FIGURES ...................................................................................................................... v ABSTRACT ................................................................................................................................. vi 1. INTRODUCTION .................................................................................................................. 1 2. PARASITE BIOLOGY, GENE SEQUENCING, AND THE EVOLUTION OF GENUS PLASMODIUM ............................................................................................................................ 8 3. GENETIC DEFENSE MECHANISMS AGAINST MALARIA .............................................. 18 4. SICKLE CELL DISEASE (HB SS), OSTEOLOGICAL IMPLICATIONS, AND PATHOLGIES IN THE FOSSIL RECORD .......................................................................................................... 25 5. CONCLUSIONS .................................................................................................................... 39 BIBLIOGRAPHY ......................................................................................................................... 42 BIOGRAPHICAL SKETCH ........................................................................................................ 46 iv LIST OF FIGURES 1. Worldwide distribution of malaria .............................................................................................. 3 2. Malaria distribution and problem areas ...................................................................................... 5 3. Distribution of Plasmodium falciparum and the sickle cell gene ................................................ 7 4. Organization of genus Plasmodium, showing the taxonomy of mammalian parasites in relation to rodent and avian parasites .......................................................................................................... 9 5. Life cycle of Plasmodium falciparum ...................................................................................... 10 6. The three life cycles of Plasmodium ......................................................................................... 11 7. The eleven species of Plasmodium ........................................................................................... 15 8. Phylogenetic tree of Plasmodium species ................................................................................. 16 9. Classification of the beta thalassaemias ...................................................................................... 21 10. Geographic distribution of the four major haplotypes of HbS .................................................... 24 11. Repair of marrow infarcts illustrating the “bone-within-bone” appearance ................................. 29 12. Cross-section of the femur, with cellular marrow and new bone growth just under the outer cortex ............................................................................................................................................ 30 13. (A) Lateral view of step-like depressions in vertebrae. (B) Diagram of the central portion of a normal and collapsed vertebra from ischemia .................................................................................. 31 14. Radiograph of the “hair-on-end” appearance, caused by widening of the diploic space ............. 32 15. Table of conditions causing the “hair-on-end” appearance ........................................................ 33 16. Cross-section at midshaft of a femur ........................................................................................ 37 v ABSTRACT The selection of the sickle cell trait occurred prior to the origin of agriculture, and possibly prior to the origin of Homo sapiens. This is shown by examining the evolutionary history of Plasmodium, the genetics of abnormal hemoglobin, and finally the skeletal traits of bone affected by sickle cell disease. Malarial parasites, particularly Plasmodium falciparum, evolved eight to ten million years ago, making it possible for humans to be infected with malaria as early as the time of the split between human and chimpanzee. A single point mutation in DNA transcription led to the circulation of hemoglobin S (HbS) in the gene pool, giving rise to a number of individuals homozygous for the trait. Individuals homozygous for the sickle cell trait (HbSS) exhibit signs of the disease in the skeleton. Traits of sickle cell disease mimic other forms of anemia, making differential diagnosis a primary goal in determining whether or not sickle cell disease is present in the fossil record. A diagnosis of sickle cell disease in the fossil record confirms the hypothesis that the sickle cell trait evolved prior to the origin of agriculture. vi CHAPTER 1 INTRODUCTION The sickle cell gene (HbS) sometimes acts as part of a genetic defense mechanism that, when heterozygous, confers resistance against malaria. The general consensus in the current literature on sickle cell disease assumes that the selection for the sickle cell gene (HbS) occurred no earlier than the time of agriculture (Livingstone, 1958, Eaton, 1994, Sherman, 1998). This thesis will challenge this hypothesis, arguing that the single point mutation causing the sickling disorder occurred prior to the origin of agriculture, and possibly before the evolution of Homo sapiens. In order to support this argument, several steps are taken in order to first understand the emergence of primate malaria, the subsequent selection of the sickle cell trait (the causal gene for the homozygous condition known as sickle cell disease), and finally the skeletal signature of this disease that is left in the hominid fossil record. Strong evidence exists which shows that the selection for the sickle cell gene occurred sometime after the origin of primate malaria, but well before the origin of agriculture. With that in mind, the age of the sickle cell trait may best be understood with regard to the presence and distribution of malarial parasites. This thesis will provide a critical review and synthesis of the literature surrounding malaria, the molecular biology and evolution of the Plasmodium species, as well as the various skeletal changes that accompany sickle cell disease. The combined epidemiological and genetic evidence for the presence of primate malaria parasites as early as 10 million years ago will support the final stages of this argument, which is to use modern skeletal markers and diagnostic features of sickle cell disease to identify possible cases of sickle cell disease in early Homo. Steinbock’s (1976) guidelines on paleopathological diagnosis and interpretation are vital to the conclusions of this paper. For the past 25 years, research has begun to focus on natural genetic defense mechanisms in hopes of creating a vaccine to cure epidemics of malaria. Malaria infects close to 500 million people a year, killing close to three million of those infected. Both humans and many other primates, including our closest relative the chimpanzee, contract malaria from the bite of a Plasmodium infected mosquito. Parasites enter the liver, and later invade red blood cells, causing them to rupture. The toxins released from the parasites into the bloodstream cause the individual to feel sick. Symptoms of malaria are felt anywhere from ten days to four weeks after infection (Hommel, 1999). Symptoms include fever, chills, 1 nausea, headache and exhaustion. If left untreated, malaria can cause kidney failure, coma and death. It is now known that there are various natural forms of resistance that have evolved as balanced polymorphisms to the deadly malaria infection. Although these will be briefly described, the focus of the present thesis is on the mutant form of the hemoglobin (HbS) gene. One copy of the HbS gene confers resistance to the Plasmodium parasite; however, individuals homozygous for the HbS trait are born with sickle cell disease. Physical anthropology offers a unique, although sometimes limited, contribution to dating the origin of sickle cell disease. Sickle cell disease leaves a specific pattern that is found primarily in the long bones and vertebra (although it has also been documented in the calvaria and pelvis). The use of modern medical techniques for diagnosing sickle cell disease can prove to be of serious diagnostic value in the study of paleopathology. A diagnosis of sickle cell disease in early Homo erectus would clarify and defend the antiquity of malaria. Additionally, an accurate determination of the origin of the sickle cell trait (HbS) and its associated varieties discussed in this thesis lends extensive insight into the human capacity to develop effective protection against malaria, and provides relevant facts and concepts regarding the design and trial of malaria vaccines. Pinpointing the birth of the sickle cell gene, as well as the first cases of sickle cell disease represented in the hominid fossil record, is an extremely important and daunting task for members of the scientific community. New techniques in gene sequencing provide important information for studying the evolution of the Plasmodium species responsible for malaria infection. In order to understand the evolutionary path this gene has taken, we must take a look at the disease it fights. Malaria is an infectious disease transmitted primarily by the Anopheles mosquito. It is caused by minute parasitic protozoa of the genus Plasmodium, of which there are over one-hundred species (Hommel, 1999). Scientists have yet to create a vaccine to cure malaria. See Figures 1 and 2 for the worldwide distribution of malaria (Trigg & Kondrachine, 1998). At present, 90 countries are known to be malarious. Almost half of those are located in subSaharan Africa. There is a great deal of literature tracing the historical documentation of possible cases of malaria. The earliest documentation is of the archaeological remains of Egyptian mummies, which exhibited enlarged spleens, an indicator of malaria, dating to more than 3,000 years ago (Sherman, 1998). Further analysis of the Egyptian mummies showed a malaria antigen in the skin and lung tissue of 2 the specimens (Sherman, 1998). Written documents have also been discovered, describing various symptoms characteristic of malaria, such as the Ebers Papyrus, which mentions splenomegaly, fever and the various curative remedies. Clay tablets found in the library of Ashurbanipal also describe deadly seasonal fevers (Sherman, 1998:3). Historical evidence indicates that the region between the Tigris and Euphrates was malarious at least 4,000 years ago. Not much was known about the biology of malaria until the 1850’s when the discovery of microbes by Louis Pasteur led the way to a much greater understanding of its pathogenesis. Perhaps the two most important finds were the discovery of the parasite in blood, and the discovery of transmission via mosquitoes. In 1879, a rod-shaped bacterium was discovered from the mud of a malarious swamp, as well as in the urine of a malaria patient (Sherman, 1998). The bacterium was identified as Bacillus malariae; however, attempts to cultivate B. malariae in the blood of infected individuals failed. The focus then shifted from bacteria to the dark red pigment of malarious blood as the cause of the disease (Sherman, 1998). Figure 1. Worldwide distribution of malaria (Trigg and Kondrachine, 1998: 12). 3 Due to the accumulations of a reddish black pigment in the spleen and liver of cadaveric material, it was proposed that the pigment itself, called hemozoin, was the actual cause of the disease. Nevertheless, in 1880, Charles Laveran microscopically examined the blood of a soldier, noticing “several transparent, mobile filaments emerging from a clear spherical body” (Sherman, 1998:5). Within one year, four different forms of the parasite had been observed, and all of the life stages of P. falciparum were recorded. Although initially met with skepticism, Laveran’s hypothesis that the disease that plagued so many was not a bacterium, but a parasite, was finally accepted. Four years later, another parasite was discovered, the second species infective to man, and was named Plasmodium malariae (Sherman, 1998). For some time, it had been thought that malaria was transmitted by the ingestion of the parasite. In 1898, Ronald Ross began to study various species of mosquitoes that fed on the blood of malaria patients. Soon he turned his attention to the brown mosquito, Anopheles gambiae, in the stomach of which he observed a black pigment. It was thereafter understood that infection by the Plasmodium parasite was transmitted by the Anopheles mosquito. Within the first decade of the twentieth century, three different human malaria parasites had been identified and described in all life stages (P. falciparum, P. vivax, P. malariae) (Sherman, 1998). The life stages, or developmental cycle, of the Plasmodium parasite is varied and complex. It took nearly forty years after the discovery of P. falciparum to determine that there are two major life cycles, or phases of development. Some parasitic stages develop outside the red blood cells. This type of development is referred to as exoerythrocytic (e-e) (Sherman, 1998). In others, especially in bird malarias, the parasite develops within the tissue and reproduces asexually. In either case, most human and primate malarias have a single e-e developmental cycle, whereas other species of Plasmodium infecting both humans and non-human primates (P. cynomolgi, P. vivax and P. simiovale) rest in the liver, and when reactivated, cause a relapse cycle (Sherman, 1998). It can be concluded that malaria is caused by the transmission of parasitic protozoa of the genus Plasmodium to the blood of the human host via the Anopheles mosquito. Currently, there are 125 species of Plasmodium (Hommel, 1999). Each species of genus Plasmodium is successful in surviving and escaping the biological defenses of their hosts, and is thus able to adapt easily (Hommel, 1999). Until recently, there has only been speculative evidence with regard to the age, or time of origin, of the 4 5 Figure 2. Malaria Distribution and problem areas (Trigg and Kondrachine, 1998: 12). endemic disease. With the help of new procedures in molecular biology and genetics, we have furthered our understanding of many of the Plasmodium species, their life cycle, and both natural and synthetic forms of resistance against the parasite. In order to study or hypothesize about forms of resistance against a disease, the evolution of that disease must be studied carefully. As was stated earlier, most scientists believe that malaria did not become a major human affliction until 10,000 years ago (Sherman, 1998, Eaton, 1994, Livingstone, 1958). It is assumed that increased sedentism as well as the environmental changes associated with the onset of agriculture were the culprits for the spread and intensity of malaria. Although this is a reasonably based claim with a good deal of merit, it is quite possible, even likely, that strains of human malaria have been evolving for millions of years. The thesis addresses the most recent evidence in genetic sequencing that claims P. falciparum is more closely related to P. reichenowi, the chimpanzee parasite, than any other Plasmodium species. On the basis of genetic sequences, the time of divergence between P. falciparum and P. reichenowi is estimated at 8-10 million years ago (Escalante and Ayala, 1996). It should be noted that the theories of Livingstone (1958) and Sherman (1998) are supported by different genetic studies claiming that P. falciparum is a recent human parasite, acquired by a host switch from birds as recently as the onset of agriculture (Waters, Higgins, and McCutchan, 1991). In either case, it follows that the various forms of natural resistance against malaria have also been evolving for multiple millennia. Figure 3 shows the distribution of falciparum malaria and the sickle cell gene (Embury, 1994). Livingstone (1958) claims that the development of the endemic P. falciparum was basically the result of iron tools and slash-and-burn agriculture. In other words, the rapid change in life style around the time of agriculture, accompanied by the decrease of tropical rainforest, directly led to the development of P. falciparum (Livingstone, 1958). This theory assumes that the removal of trees and brush would have led to an increase in standing water and sunlight, which are the necessary conditions for the mosquito vector, Anopheles gambiae to breed. Although the effect of material culture on the evolution and spread of this parasite is undoubtedly major, the roles of sedentism and agriculture are not entirely able to explain this phenomenon. As a matter of fact, the currently accepted notion that agriculture triggered epidemic malaria has yet to be 6 Figure 3. Distribution of (top) Plasmodium falciparum and (bottom) the sickle cell gene (Embry, 1994:15). critically examined. While the theory that agriculture triggered epidemic malaria may help to explain the distribution and further spread of malaria, it does not entirely explain the origin or first cases of malaria, much less the selection of the sickle cell trait (HbS), or other natural defense mechanisms against malaria infection. In other words, the original Livingstone (1958) hypothesis, versus the claims made in this thesis, are not mutually exclusive. Rather, the currently held hypothesis provides a need for a deeper understanding of the antiquity of natural defense mechanisms against malaria, namely the sickle cell gene. The next chapter offers sound evidence that human and other primate malarial parasites have existed for millions of years, which at the very least illustrates that primates and hominids were able to contract malaria millions of years prior to sedentary groups of Homo sapiens and slash and burn agriculture. 7 CHAPTER 2 PARASITE BIOLOGY, GENE SEQUENCING AND THE EVOLUTION OF GENUS PLASMODIUM As noted, there are over 125 species of Plasmodium, infecting a number of different vertebrates. Plasmodium is the only genus in the suborder Haemosporina. The genus Plasmodium generally infects a variety of reptiles, birds and mammals. Plasmodium species are lower eukaryotes with a genetic complexity five times greater than that of bacteria (Gilles, 1999). All of the species of this suborder are obligate intracellular parasites for almost all their life cycles and have two hosts: a vertebrate host, in which reproduction is asexual (also called the intermediate host), and an invertebrate host, blood-sucking diptherous insect (called the definitive host), in which fertilization occurs (Gilles, 1999: 26). The complexity of Plasmodium life cycles, combined with the considerable polymorphism of the organism, facilitates clever ways in adapting to changing situations. The Plasmodium parasite has the uncanny ability to choose, or navigate its path during different stages of its biological development. The genus of Plasmodium is divided into 10 subgenera (shown in FIG 4) (Gilles, 1999:27). Human and primate malaria are all included in either the subgenera Plasmodium or Laverania, while all other mammalian parasites are confined to the subgenus Vinckeia. In general, four species of Plasmodium infect humans: P. falciparum, P. vivax, P. ovale, and P. malariae. The classification used to differentiate these species is largely based on morphological characteristics and certain features of the life cycle. Morphological criteria used in the classification of Haemosporina was developed by Garnham (1966), which included the shape of the trophozoite, the gametocyte and the oocyst, the number of nuclei in the erythrocytic and exoerythrocytic-erythrocytic schizonts, the aspect and distribution of the pigment and the nature of the damage induced by the parasite in the host cell. Issues related to taxonomic status are important in phylogenetic studies. For example, the analysis of sequence 8 Figure 4. Organization of genus Plasmodium, showing the taxonomy of mammalian parasites in relation to rodent and avian parasites (Gilles, 1999:26). homologies of the circumsporozoite protein (CSP-1) gene and the comparison of small subunit ribosomal RNA genes have provided new information, such as the segregation of murine (or rodent) plasmodia from other mammalian parasites. Studies of ribosomal RNA and CSP-1 sequences, show that between 8-10 million years ago, Plasmodium parasites originated that would later be infective to the Homo species (Escalante and Ayala, 1996). The conclusions of this important study are central to this thesis. As stated earlier, all of the malaria species undergo a complex developmental cycle, with a sexual cycle completed in the invertebrate host (Anopheles mosquito) and an asexual cycle in the vertebrate host. Figures 5 and 6 diagram the sexual and asexual cycles of Plasmodium (Gilles, 1999: 29), ( Sherman, 1998:9). Sexual Stage The sexual stage of the parasite is a complex process of differentiation ordered so that fertilization will take place within the mosquito midgut. It is important to note that the zygote/ookinete is 9 Figure 5. Life cylce of Plasmodium falciparum (Gilles, 1999;29). the only part of the life cycle when the organism is diploid and when meiosis takes place (Sinden, 1998). Once the female Anopheles mosquito takes up malarial gametocytes through a blood meal, a sequence of events takes place, where the gametocyte is fertilized, forms into a zygote, and is transformed into an ookinete. The ookinete travels to a special receptor on the midgut epithelial cell of the Anopheles, where it attaches itself and differentiates into an oocyst. The oocyst is the encysted form of the fertilized macrogamete, or zygote. After the oocyst completes its maturation, it ruptures and releases sporozoites into the haemocoel, or body cavity, of the mosquito. The sporozoites are small, active, usually elongate, sickle-shaped spores that will be transferred into the host from the vector. The sporozoites will travel through the haemocoel of the mosquito, making their way to the salivary glands. During the migration from the oocyst to the salivary glands, the sporozoites themselves fully mature, including developing the quality of their surfaces with the circumsporozoite protein (CSP-1). The sporozoites then collect in the salivary glands before finally crossing a chitinous wall into the actual salivary duct, where they await 10 Figure 6. The three life cycles of Plasmodium (Sherman, 1998:9). transmission. How the sporozoite survives in the haemocoel of the Anopheles mosquito without being killed by the destructive haemolymph, how it finds its way to the salivary glands, and how it crosses the chitinous wall to the salivary duct are still largely unknown (Hommel, 1999). Asexual Stage When a sporozoite is introduced into human skin via the Anopheles mosquito, it circulates in the bloodstream for approximately 10-60 minutes before moving into the liver, where it then invades hepatocytes, or liver cells. (Sherman, 1998). When the sporozoite enters the hepatocyte, it undergoes changes in morphology, including the creation of a vacuole in the hepatocyte. Within this vacuole, the nucleus of the sporozoite divides many times, causing the mass of the cytoplasm to increase dramatically. The actual number of nucleotic divisions and duration of schizogeny varies from one species to another. The rate at which a sporozoite is able to induce an infection is also speciesdependent. P. falciparum and P. vivax, for example, are considered to be the most infective because 11 they are able to infect with only 10 sporozoites (Hommel 1999:29). After the given amount of nucleotic divisions, the cytoplasm segments and individual merozoites are created. This entire process, from the invasion of the hepatocyte to the splitting of the cytoplasm, is referred to as blood schizogeny in the literature. This specific process is referred to as exoerythrocytic blood schizogeny, because it takes place outside the erythrocyte, within a vacuole in the hepatocyte. The next stage of asexual development in the vertebrate host involves the merozoites that were formed when the cytoplasm of the hepatocytes ruptured. This stage is referred to as erythrocytic schizogeny. The merozoite, when first formed, is an oval cell and is equipped with special invasion organelles, such as a special surface coat and an apical complex at the tip. The purpose of the merozoite is to invade the red blood cell, where it then loses its invasive features and transforms into a round trophozoite in the cytoplasm of the erythrocyte. The trophozoite continues to grow, just as the sporozoites grow in the exoerythrocytic phase. Once fully grown, the merozoite undergoes a number of nucleotic divisions and is eventually released to travel through the blood and invade other erythrocytes. The merozoites released at the end of schizogonic development have a short life span, and can only invade erythrocytes. Once they invade, they can either undergo another cycle of schizogonic development, or differentiate into a gametocyte. When viewed microscopically, the development of the parasite will result in several unique morphological changes seen in the membrane of the erythrocyte. Some of these features are species specific and are thus useful in taxonomic classification. Plasmodium species, whether in the sexual or asexual stage of development, spend most of their existence as intracellular organisms. The sexual and asexual stages described above are specific to Plasmodium falciparum infecting humans. There are slight variations of these stages between different primate and avian species. Nonhuman primates have been widely used for malaria studies because of the close resemblance of their host-parasite relationship (Gysin, 1998). Plasmodium parasites of nonhuman primates are morphologically very similar and may share a close phylogenetic relationship. In fact, it has been noted that many of the species infecting chimpanzees, monkeys and man may be biological variants of just one species (Hommel, 1999). The study of life cycles and reproduction in the genus Plasmodium is essential to the study of interspecies relatedness, and with the help of molecular biology, for making correct assumptions about the age of a given species. 12 A solid understanding of the morphological and biological characteristics of Plasmodium species is necessary for taxonomic and evolutionary studies of endemic malaria. The genetic make-up of the Plasmodium parasite also lends important information to the understanding of its evolutionary history. Nucleic acids and amino acids are sometimes referred to as informational macromolecules because they carry the information that codes for how an organism develops and how it functions (Escalante and Ayala 1997). This genetic code also retains a record of the organism’s evolutionary history. The evolutionary history of Plasmodium makes it possible to reconstruct the evolutionary relationships of parentage, but most importantly, it makes it possible to time those events in a species’ history (Escalante and Ayala 1997:22). Evolution in Plasmodium, as well as most other organisms, occurs by the substitution of the nucleic and amino acids, one at a time so that “the number of differences between two organisms is an indication of the recency of their common ancestry”( Escalante & Ayala 1997:21). Escalante and Ayala (1997) state that there are three notable advantages of molecular biology over paleontology and comparative anatomy for making an accurate interpretation of evolutionary history. First, the information is readily quantifiable, such as the number of units determined whenever the sequence of the component units is known for a given gene. Another advantage is that most organisms can be compared by matching homologous macromolecules, no matter how different or how difficult it may be to compare them by other means. The final, and perhaps most important, advantage that molecular biology offers is that the results obtained by the study of one gene or protein can be used as a “phylogenetic hypothesis to be tested by examining other genes or proteins” (Escalante & Ayala 1997:23). The phylogeny of the phylum Apicomplexa has always been the subject of controversy and frequent revision. Even more controversy is attributed to discussions of the class Hematozea within the phylum Apcicomplexa, to which the genus Plasmodium belongs. Because Plasmodium parasites are responsible for millions of deaths a year, a great deal of research is conducted in hopes of understanding how to eradicate, or at least suppress the lethal affects of malaria infection. An accurate understanding of the taxonomy and phylogenetic history of genus Plasmodium may yield economic and medical consequences. Within the past decades of genetic research, there have been several conflicting interpretations of the evolutionary history of genus 13 Plasmodium (Livingstone, 1958; Hoeprich, 1989; Waters, Higgins, and McCutchan, 1991). What follows is the summary of an analysis by Escalante and Ayala (1994, 1997) which is the only study which states that Plasmodium falciparum is more closely related to Plasmodium reichenowi (host Pan troglodytes) than to any other species within the genus. The evidence presented by Escalante and Ayala (1997) is central to the argument that sickle cell trait was selected for much earlier than currently thought. Escalante and Ayala (1994) examined two aspects of Plasmodium, the SSU rRNA (small subunit ribosomal RNA) genes as well as the CSP (circumsporozoite protein) gene. The CSP gene codes for a protein that is particularly useful because it is part of the apical complex, or the tip of the parasite that codes for the strength of its infectivity. The CSP gene has been used extensively as a target for vaccine development (Rich, Hudson & Ayala, 1997). The success of such efforts determines the variation, or, the level of diversity for the CSP gene. Although there are four species that are currently considered parasitic to humans (P. malariae, P. ovale, P. vivax, and P. falciparum), P. falciparum parasites are known for their particular virulence in humans. This has been generally attributed to the notion that Plasmodium is a recent human parasite. This recency can be explained by a host switch (from domestic birds to humans) as recently as the onset of agriculture some 8,000-10,000 years ago. If true, then Plasmodium species infective to birds are phylogenetically more closely related to Plasmodium species infective to humans than any other of the Plasmodium species that are infective to primates. The date for the recent common ancestor of Plasmodium falciparum is estimated from the analysis of SSU rRNA genes. The methodology used in most of the studies of P. falciparum is the examination of only those genes that are expressed during the sexual stage in the vector (Waters, Syn and McCutchan, 1989). The sexual stage studies of P. falciparum produce a much earlier date for the recent common ancestor, just prior to the origins of agriculture. Recently, there has been argument that the population structure of Plasmodium falciparum is clonal, which focuses much more molecular research on the asexual phase of the parasite’s development (Rich, Hudson and Ayala, 1997). Using a variety of statistical comparisons, the results of these asexual stage studies, produce a much older date for the most recent common ancestor. 14 In either case, every study on the evolutionary history of Plasmodium addresses the same important question, its date of origin. According to Escalante and Ayala (1996), an examination of genetic sequences that occur during the asexual phase of the parasite yields a more accurate understanding of the evolutionary path of the genus. Escalante and Ayala (1996) analyzed the SSU r RNA genes of eleven Plasmodium species to reconstruct the phylogeny of the genus. Figure 7 lists the eleven species as well as their known hosts and geographic distribution (Escalante and Ayala, 1994). The phylogenetic relationships between Plasmodium species were determined by two different statistical methods, neighbor joining and maximum likelihood. First, the genetic sequences were aligned using the CLUSTAL-V program, a statistical measure used to construct phylogenetic relationships. The alignment of the sequences produced a reliable alignment for 1,620 base pairs (Escalante & Ayala, 1996). The reliability of the tree construction was assessed by the bootstrap method. After applying the neighbor joining and maximum likelihood equations, as well as applying the bootstrap method, a phylogenetic tree was composed for the eleven species (see Figure 8) (Escalante and Ayala, 1994). The numbers at the tip of each branch are the “bootstrap values that indicate the percentage of times (out of 1,000 replications) that the set of species in the cluster to the right of the branch appeared as a monophyletic clade” (Escalante & Ayala 1996:25). In other words, the branch lengths reflect the genetic distance between each species. The Figure 7. The eleven species of Plasmodium (Escalante and Ayala, 1994: 23). 15 Figure 8. Phylogenetic tree of Plasmodium species (Escalante and Ayala, 1994: 24). most important conclusion to be drawn from this analysis is that P. falciparum clusters with P. reichenowi, the chimpanzee parasite, with a bootstrap reliability of 100%, and that P. vivax, another human parasite, clusters with the three monkey parasites (P. fragile, P. knowlesi, and P. cynomolgi). Therefore, the authors conclude that P. falciparum is more closely related to P. reichenowi than any other Plasmodium species. In addition, the rRNA genes show that the estimated time of divergence between the two Plasmodium species is 8-10 million years ago (Escalante and Ayala, 1994). Although the time of divergence between P. falciparum and P. reichenowi is estimated to have arisen prior to that of the host species, (human and chimpanzee), it does indicate that the two different Plasmodium species were present by the time of the host split. The conclusive interpretation made from this genetic study is that “P. falciparum is an ancient human parasite, associated with our ancestors since the divergence of the hominids from the great apes” (Escalante and Ayala, 1996: 26). Furthermore, the other human parasites, P. malariae, P. vivax and P. ovale, are very remotely related to P. falciparum and P. reichenowi, which implies that the evolutionary divergence of these parasites greatly predates the origin of hominids. This statement is consistent with the physiological and epidemiological characteristics of P. falciparum, P. vivax, and P. malariae (Lopez-Antunano and Schumunis 1993, Escalante and Ayala, 1996). 16 The results of this molecular research substantiate the claim that malaria parasites infective to humans and chimpanzee are indeed ancient enough to have plagued hominids in Africa beginning from at least the time chimpanzees and hominids diverged from the common ancestor. Once this has been established, it is necessary to consider the principles of natural selection. If a significant number of hominids were infected with malaria parasites, it follows that any genetic mutation that confers resistance to the virulence of the infection would have been selected. The rest of this thesis reviews the various genetic defense mechanisms that have arisen, which confer some resistance to malarial infection, and argues that the selection for the sickle cell trait (HbS) occurred at least as early as the origin of Homo erectus. 17 CHAPTER 3 GENETIC DEFENSE MECHANISMS AGAINST MALARIA INFECTION The date of origin for hominid Plasmodium parasites established by Escalante and Ayala (1994, 1996) is extremely important to the argument that sickle cell disease, and associated hemoglobin pathologies, are of ancient origin. It is known that the sickling characteristic of abnormal hemoglobin protects individuals heterozygous for the trait from the deadly effects of malaria by inhibiting the reproduction of the parasite (Eaton 1994, Serjeant 1992, Mankadd and Hoff, 1992). It is thought that the Plasmodium parasites are unable to survive because there is a lower available oxygen supply caused by abnormal hemoglobin. It has also been shown that there is a direct correlation between the frequency of Plasmodium falciparum prevalence throughout the world and the level of individuals heterozygous for the sickle hemoglobin gene (HbS). The actual process of hemoglobin synthesis is very complex and finely balanced, where, unfortunately, many errors can occur. The structural variants of hemoglobin illustrate very well the patterns of abnormality (Serjeant, 1992). Because there are over two hundred variants of hemoglobin, it is logical to conclude that mutations generated by various substitutions in nucleotide sequences coding for hemoglobin have been evolving for quite some time. This chapter examines the genetics of variant forms of hemoglobin, with particular emphasis on the sickle cell trait (hemoglobin variant S). Many of the sickling forms of hemoglobin offer resistance to the malarial parasite and, due to the current frequency of the gene in areas of endemic malarial infection, it is argued here that the sickle cell gene emerged prior to the evolution of Homo sapiens. Hemoglobin is a molecule carried by erythrocytes that picks up oxygen in the lungs and delivers it to the entire body. In order for hemoglobin to function properly, two proteins must form a bond. One of the proteins is known as the alpha and the other, beta. Segments within DNA that code for these particular proteins make up the genes for hemoglobin. The phenotypic expression of the alpha chain of proteins is under the control of a duplicated pair of genes on chromosome 16, whereas the beta chain, controlled by only a single pair of genes, is coded for on chromosome 11 (Serjeant, 1992). Normal coding of hemoglobin within the alpha and beta proteins results in the formation of normal red blood cells that maintain a disc shape. Under these normal conditions, the hemoglobin molecules exist as 18 single, isolated units in the red cell. When a variant form of hemoglobin is coded for in transcription, the hemoglobin will begin to form long chains, or polymers, within the red cell. These polymers distort the red cell and cause it to bend out of shape. The sickle hemoglobin will undergo several phases of polymerization and depolymerization between circulation to and from the lungs. This repeating process eventually damages the hemoglobin and leads to the destruction of the erythrocyte. Of the many variants of hemoglobin that have evolved over time, the most common mutation is due to single base substitution. A single base substitution is one in which only one nucleotide is changed, such as from purine to pyrimidine (Mankad and Hoff, 1992). In the case of mutant hemoglobin S, there is a single base substitution in the codon for the sixth amino acid in the beta globin gene, which substitutes the amino acid valine for the normal glutamic acid (from GTG to GAG) (Mankad and Hoff, 1992). There are, however, several other ways abnormal hemoglobin is formed. Double base substitution occurs when two separate base changes result in two amino acid substitutions in the same chain. All of the forms involved in double base substitution include the substitution for HbS, therefore they manifest sickling. Aside from the single and double base substitutions that occur in the middle of a nucleotide sequence, there may also be mutations affecting the STOP codons (UAA, UAG, UGA) (Serjeant, 1992). STOP codons dictate the termination of globin synthesis, therefore when a mutation occurs in a STOP codon, the mRNA will continue to read until another STOP codon is reached. In other words, this type of mutation will lead to an elongated hemoglobin chain. STOP codon mutations usually produce thalassemia-like conditions described below. Despite the various ways in which mutant hemoglobin arises, whether it is from single or double base substitutions, or STOP codon substitution, all lead to an array of conditions in which the pathology may be attributed to sickle hemoglobin. The most prevalent genotypic expression of sickle hemoglobin is hemoglobin S (HbS) described above. Another less common form of sickle hemoglobin is hemoglobin C (HbC), which results in an amino acid substitution at the same site in the beta chain as HbS. In this specific case, the first nucleotide in the codon determining the sixth position in the amino acid changes from a G to an A, reading GAG to AAG. This form inserts lysine in place of glutamic acid, instead of valine in the case of HbS. Most of the other well known variations, such as HbD and HbO also arise from a single point mutation. However, these variations occur at different loci on the beta chain of the hemoglobin. For example, HbD, also known as 19 Punjab hemoglobin, results in the substitution of glutamine for glutamic acid (CAG to GAG) at position 121 on the beta chain. Hemoglobin O, or Arab hemoglobin, substitutes lysine for glutamic acid, similar to variant HbC, except it occurs at the same locus on the beta chain as HbD, position 121 (Nagel, 1994). Thalassaemias are closely related to the variant forms of hemoglobin, although the pathological condition is slightly different, and in many cases more severe than that of sickle hemoglobin. Although there are many different types of thalassaemia, this paper will focus alpha thalassaemia and beta thalassaemia. Thalassaemias are characterized by globin-chain imbalance, due to the coding for inadequate chain synthesis. It is possible for the sickle HbS hemoglobin to be inherited in combination with alpha thalassaemia, since the HbS mutation occurs on the beta chain, and the thalassaemia is the result of a mutant alpha chain (Serjeant, 1992:11). Alpha thalassaemias usually result from some sort of gene deletion, where a pair of alpha globin genes is deleted during transcription. Other forms of alpha thalassaemia are due to deletions at a splicing site or in the STOP codon. Beta thalassaemias, on the other hand, are the result of several different single point mutations (Kazazian, 1990). Figure 9 is a classification of the major types of beta thalassaemias, also describing the ratios of variant hemoglobin and the position where mutations occur (Serjeant 1992: 389). The effects of beta thalassaemia on overall gene function are more clearly seen when other areas of the beta globin chain are abnormal. All of the forms of beta thalassaemia arise in a very similar way to that of the variant hemoglobins described above. Most forms arise with the single point mutation of a certain position within the sequence of amino acids in the hemoglobin gene. Taking this into consideration, it can be said that the characteristics and expression of all mutant forms of hemoglobin depend on the extent of the deletion or substitution with particular importance on the position or location of substitution, as well as the function of the remaining genes. One of the major interests of epidemiologists is to understand and explain the frequency as well as distribution of the various forms of hemoglobin mutation. In most cases, individuals heterozygous for a mutant form of hemoglobin will be relatively asymptomatic and may be afforded a certain degree of protection against Plasmodium falciparum. The geographic distribution and frequency of mutant hemoglobin genes is extremely helpful in the piecing together of an evolutionary history for hemoglobin 20 Figure 9. Classification of the beta thalassaemias (Serjeant, 1992: 389). mutation. What follows is a brief discussion on the most prevalent forms of these genetic abnormalities according to their geographic distribution. Hemoglobin S (HbS), also referred to simply as sickle hemoglobin, has commonly been misconceived as occurring solely among African and African-American populations (Serjeant, 1992). In fact, the HbS gene is widely distributed among peoples in Italy, northern Greece, southern Turkey, the Eastern Province of Saudi Arabia, and India. Hemoglobin C, less widely distributed, is primarily found in Ghana and Burkina Fasso. It has also been found that there are genetic isolates in parts of northern Israel, which may represent an entirely new mutation (Rachmilewitz et al, 1974). Hemoglobin D, also known as Punjab, is a widely distributed yet lower frequency mutant hemoglobin. It reaches its highest prevalence among the Sikhs in India, although it has also been documented in black populations in both the Caribbean and North America. Hemoglobin O, named Arab hemoglobin, is rare and has lower distribution and frequency rates than other mutant hemoglobin. Hemoglobin O has been recognized in Israel, Sudan, Kenya, Jamaica, Bulgaria and the United States (Serjeant, 1992). 21 The distribution and frequency of the thalassaemia genes vary a great deal. It can be said that one form of alpha thalassaemia occurs in nearly five percent of all peoples in South East Asia. This form, referred to as alpha negative (á-), is extremely rare in African populations. On the other hand, alpha positive thalassaemia (á+), occurs in twenty four to thirty five percent of African-American populations when both heterozygote and homozygote frequencies are combined. Beta thalassaemia genes follow a similar, yet contrasting distributional pattern to those of the alpha thalassaemias. The beta negative (â-) thalassaemia genes are found in higher numbers among North Africans and Jamaicans of West African origin. On the other hand, beta positive thalassaemia (â+) genes predominate in Turkey as well as in parts of Saudi Arabia (Serjeant, 1992). Studies that assess the geographic distribution and frequency of hemoglobin mutation attempt to explain the origin and spread of genetic change. Epidemiologists find it particularly interesting that there is a correlation between frequency of malaria infection and the prevalence of the sickle cell trait, among other abnormal hemoglobins, in concentrated areas of Africa. The mutant hemoglobins described above, including Hemoglobin S, C, D, and O, as well as the alpha and beta thalassaemias, all contribute in varying degrees to the retardation of the growth of P. falciparum (Hill & Weatherall, 1998). When compared, the sickle cell trait (HbS) offers more resistance to the deadly effects of P. falciparum than do some of the other forms of genetic defense. Therefore, any attempt to understand the current distribution of Hemoglobin S, or any other mutant hemoglobins depends heavily on the time and place of origin for those mutations. This thesis focuses primarily on the abnormal Hemoglobin S, and especially on the homozygous condition of sickle cell disease. Nevertheless, it is helpful to understand the variety of abnormal hemoglobins as described above, as well as to observe the geographic distribution and frequency of each of these genetic mutations. Throughout the years of research on sickle cell disease, there have been many explanations for where, when and how the sickle cell gene arose. Substantial evidence now suggests that the sickle cell mutation occurred as several independent events (Serjeant, 1992, Mankad & Hoff, 1992). For many years, however, the single mutation theory of sickle cell evolution was held as a very likely possibility. Lehmann (1954) proposed that a single mutation in hemoglobin occurred in Neolithic times. Based on modern frequencies of the gene, it was assumed that the mutation originated 22 in the Arabian peninsula. If this were true, then it would follow that changing climatic conditions correlated with a migration of peoples into India, Eastern Saudi Arabia and into Equatorial Africa. Later, it was found that the distribution of certain agricultural practices, in addition to the geographical distribution of the sickle cell gene supported the single origin and migration hypothesis because the gene frequency declined from East to West Africa (Mankad & Hoff, 1992). In addition, there were slightly higher levels of HbS gene in the north compared to the area south of the Zambesi River, which is compatible with the idea that the river acted as a barrier for southern migration. This hypothesis raises some interesting questions about the effects of climate as well as geomorphologic determinants in gene distribution. However, modern gene frequency and distribution analysis alone can’t be the sole determinant of the genes’ origin. Studies in molecular genetics support another theory for the evolution of the sickle cell trait. Specifically, the recognition that there are many variations in DNA structure, or polymorphisms, and that they can be used as genetic markers implies that the sickle cell gene, consisting of many different variations, was the result of multiple independent mutations. The multiple mutation theory is based primarily on an analysis of beta-globin haplotypes, and the geographic distribution of the different beta haplotypes throughout Africa. The beta chains of hemoglobin consist of a number of different enzymes which identify multiple recognition sites. Determining the pattern of these polymorphic sites is essential in estimating the length of time a particular mutation has existed. Using the beta globin enzymes to identify various chromosome structures showed that there are four principal beta globin haplotypes in Africa, all of which are polymorphisms of the hemoglobin S. The four major â haplotypes are classified as the Benin, Bantu, Senegal and Asian or Indian haplotypes. Figure 10 (Mankad & Hoff, 1992) is a map showing the geographic distribution of the four major haplotypes of HbS. It can be concluded that the combination enzyme analysis identifying the four major haplotypes and spatial distribution of the different â haplotypes show strong support for a multiple mutation origin of the sickle cell gene. Furthermore, the variation of haplotypes and geographic distribution can be used to calculate when the gene first occurred. This process is similar to that discussed in Chapter 2 for calculating the date of origin for Plasmodium species. Kurnit (1979) attempted to estimate the date of origin of the sickle cell gene by using statistical measurements, which resulted in a date of origin between 23 Figure 10. Geographic distribution of the four major haplotypes of HbS (Serjeant, 1992: 18). 70,000 and 150,000 years ago. Historically it has been assumed that the age of the HbS mutation is coincident with the time malaria became endemic (Nagel, 1984, Livingstone, 1958, Eaton, 1994). This assumption is based almost solely on the selective advantage against various Plasmodium strains afforded to those individuals with the heterozygous HbS gene. It is still largely held that epidemic proportions of malarial infection coincided with the time of agriculture, which assumes that malaria did not threaten people on a large scale until they became sedentary. It has, however, also been proposed that the HbS mutation could have been “lying around” for thousands of years, and that the expansion of malarial infection and the mutation should not be associated in a causal relationship (Nagel, 1984). This thesis, on the other hand proposes that malaria has plagued primates for millions of years, causing some degree of selective pressure and change within the gene pool. It also postulates that hemoglobin mutation, specifically HbS, was selected for during the time of Homo erectus. 24 CHAPTER 4 SICKLE CELL DISEASE (Hb SS), OSTEOLOGICAL IMPLICATIONS, AND PATHOLOGIES IN THE FOSSIL RECORD The inheritance of one copy of the mutant hemoglobin S affords individuals a selective advantage against malaria, and is thus found in high frequencies in geographic areas with a correspondingly high level of malaria infection. It follows that areas with high frequencies of the heterozygous genotype have high frequency of the HbS allele which also gives rise to higher levels of the homozygous genotype. If both of the beta globin genes code for HbS, the individual will suffer from what is referred to as homozygous sickle cell disease. As shown in Chapter 3, sickle hemoglobin (HbS) causes the red cells to form long chains, or polymers, which become rigid and cause the red cell to distort, giving the appearance of a crescent or sickle shape. These rigid and distorted red cells eventually fail to move through small blood vessels. Once red cells are blocked from travel, blood flow is cut off to the tissues. Repeated episodes of red cell blockage produces what is called tissue hypoxia (Diggs, 1992). Tissue hypoxia refers to an extended period of low oxygen supply. Red cells that have been depleted of oxygen will eventually die. Sickle cell disease is commonly referred to as sickle cell anemia primarily due to the process known as hemolysis, or red cell destruction. With sickle cell disease, the production of red cells increases dramatically, although they are unable to maintain a normal level due to tissue hypoxia and the resulting hemolysis of red cells. The average half-life of red cells is close to forty (40) days, although in patients with sickle cell disease, this value can fall to as low as four days (Serjeant, 1992). This chapter will examine the pathology of sickle cell disease with particular emphasis on the physiological changes to bone, in hopes of securing a strong argument for the inclusion of sickle cell disease in the list of pathologies present in the fossil record. One of the major obstacles in making this case is the fact that the survival rate for individuals homozygous for the sickle cell allele (Hb SS) is reportedly low. As the result of further and more advanced study, however, there has been increasing recognition of a wide variability of hemoglobin mutation as well as varying degrees of severity in sickle cell disease sufferers. In other words, the inheritance of homozygous sickle cell trait, and thus sickle cell 25 disease, is not the sole determinant of severity, and for that matter, of survival. Serjeant conducted the first set of extensive studies on sickle cell disease survivors, describing sixty (60) patients in Jamaica over the age of thirty (Serjeant, 1992). Not only has there been documentation of long term survivors with the homozygous condition, but there is also an increasing awareness of significantly benign cases of sickle cell disease in younger individuals (Shurafa, 1982). Although improvement in medical care has grown tremendously throughout modern history, the prognosis of individuals can’t be solely attributed to improved treatment. Serjeant (1992) notes that the awareness of a more varied spectrum of severity in sickle cell disease is not only important to general prognosis, but that it should also contribute to a clearer understanding of the pathophysiology of the disease. For the purpose of this thesis, only those aspects of sickle cell disease that involve the skeletal system will be examined, using modern radiological and clinical cases of variability. Due to chronic anemia in patients with sickle cell disease, as well as the constant impairment in the flow of blood in terminal vessels, there are progressive atrophic changes in bones throughout the body (Diggs, 1992). These atrophic changes are usually the result of marrow hyperplasia and bone infarction. Infarction is the term for localized cell death in bone marrow and adjacent bony structures, whereas marrow hyperplasia generally refers to the abnormal and continual increase in the number of red blood cells. Marrow hyperplasia is due to hypoxia resulting from the hemolysis of red blood cells. Normal development of bone marrow begins as highly hematopoietic, meaning that it is producing a large number of blood cells. Hematopoietic marrow has a red appearance, although during normal development, a child’s bone marrow will transform at a specific rate into fatty, or yellow, marrow (Brogdon et. al., 1992). The conversion from red to yellow marrow begins in the small bones of the hands and feet and follows a gradual path, transforming bone marrow from the distal to the proximal. The adult stage of bone marrow, reached around the age of twenty-five, exhibits red marrow only in the vertebrae, sternum, ribs, pelvis, skull, and proximal shafts of the femora and humeri (Brogdon et al., 1992). Interestingly, Milner et. al (1994) found that the majority of sickle cell related infarcts within the skeletal system can be seen in the “long bones, ribs, sternum, vertebrae, and pelvic bones, and occasionally in the skull and facial bones” (645). 26 When marrow hyperplasia takes place, there is a high cellular increase in red blood cells because of the need to produce cells as fast as they are destroyed. The resulting cellular increase within the marrow causes the resorption of bony trabeculae in spongy bone, and thus causes the cortex to thin (Brogden et. al., 1992). The cortex thins so that the marrow cavity can widen, attempting to make room for vital oxygenated blood cells. This process leads to a weakening of osteoporotic bone, the results of which are visible pathologies in specific areas of the body. In addition to the effects of marrow hyperplasia, it is also clear that when the cortex, as well as the medullary space, is deprived of an adequate amount of oxygenated blood, a state referred to as ischemia, a localized infarct will occur (Milner et. al., 1994). The medullary cavity within long bones is supplied by the nutrient artery, therefore, when the cavity is in a hypoxic state, it is highly susceptible to infarction. Medullary infarcts usually heal, but they may lead to marrow fibrosis or calcification (Brogdon et. al., 1992). While it is most common in skeletal findings of sickle cell disease to find changes related to marrow hyperplasia and ischemia, those changes that are more impressive are associated with bone infarction (Brogdon et. al., 1992). Bone infarction is also known as osteonecrosis, or bone death, both known to cause a separation of the periosteum from the cortical shell of the affected bone. Just as red cells increase production during hemolysis, new osteocytes begin production to counterbalance the damage of osteonecrosis. This process of new bone formation is specifically initiated by periosteal osteoblasts, thus causing the cortex to be thickened (Brogden et al., 1992) Figure 11 shows the repair of marrow infarcts illustrating “parallel lines on the medullary side of the cortex, giving the appearance of ‘bone within bone’” (Milner et al., 1994). Hershkovitz et al. (1997) examined the skeletal remains of children, looking particularly at different types of anemia and how to differentiate between the pathologies seen in sickle cell anemia. Hershkovitz found that “calvarial thickening, tibial and femoral cortical bone thickening and bowing,” are characteristic of sickle cell anemia, and that rib broadening, granular osteoporosis are associated, but not specific (Hershkovitz et al., 1997). With regard to long bones, specifically the femur, acute diaphyseal infarction occurs between the intermediate segment between the diaphysis and metaphysis at the point furthest from the nutrient artery (Serjeant, 1992). Figure 12 shows a section of the femur, with cellular marrow and new bone growth just under the outer cortex (Graham, 1924). Interestingly, a study of Nigerian patients (Bohrer, 1970), 27 shows that the femur and tibia were most frequently affected by infarction, whereas in a study of longbone infarction of American patients (Keeley and Buchanan 1982), the humerus was more frequently affected. Miler et. al (1992) note that osteonecrosis of the femoral and humeral heads are the most frequent among patients with homozygous sickle cell disease as well as those who have alpha thalassaemia. As a result, the hip and shoulder joint are equally affected. Hershkovitz notes that the metaphyseal and articular surfaces of long bones are usually free of any sign of pathological process or developmental disturbances (Hershkovitz et al., 1997). The tubular bones of the hands and feet are also adversely affected. Throughout childhood, these bones normally contain a predominance of oxygen-demanding blood. When erythrocytes sickle, hypoxia is highly likely to deprive the tubular bones of adequate oxygen, thus causing necrosis (Diggs, 1992). Necrosis of the central part the metacarpal epiphysis has shown to result in deformities, premature fusion, and shortened deformed bones. Hershkovitz claims that calcaneal and metacarpal lesions are highly characteristic of the pathology associated with sickle cell anemia (Hershkovitz et al., 1997). The vertebrae also exhibit noticeable pathological characteristics. The medullary spaces in spinal bones are also filled with red marrow, and the central portion of each vertebra is the terminal point of circulation. When hypoxemia occurs due to sickled erythrocytes, the central area of the vertebral bodies will become depressed by vertebral discs (Diggs, 1992). When viewed laterally, vertebral bodies look flattened and widened, giving a step-like impression (Milner et al., 1994). Figure 13 shows a lateral spine x-ray of a woman exhibiting the “step-like depression” caused by ischemia to the vertebral bodies as well as a diagram of the central portion of a normal and a collapsed vertebra from ischemia (Milner et al., 1994: 648). It illustrates how bone growth is inhibited due to the obstruction of the main vertebral artery during microcirculation. It is very important to note that this vertebral compression is primarily found in patients with sickle cell disease (>30%), and can aid in cases of differential diagnosis, where thalassaemia or iron-deficiency anemia need to be discounted (Steinbock, 1976). Other bones, such as those of the skull, mandible, and more rarely the ribs, sternum and clavicle are also affected by various forms of anemia. Changes in the skull can be indicative of the presence of 28 Figure 11. Repair of marrow infarcts illustrating the “bone within bone” appearance (Milner et. al., 1994: 651). 29 sickle cell disease, as well as iron deficiency anemia and thalassemia. Marrow hyperplasia results in the widening of the diploic space, usually affecting the frontal and parietal bones. The widening of the diploic space coarsens the trabeculae, which leads to a striking “hair-on-end” appearance, shown in Figure 14 (Diggs, 1992:157). Spongy hyperostosis was the first pathology attributed to a hematological disorder, such as sickle cell anemia or thalassaemia. This was first discovered when Ales Hrdlicka described lesions on the skull vaults of Peruvian Indians that were characterized by thickened areas of cortex mixed with porotic spaces, found symmetrically distributed on both sides Figure 12. Longitudinal Cross-section of the femur, with cellular marrow and new bone growth just under the outer cortex (Graham, 1924). of the parietals and on the occipital (Steinbock, 1976). At the time, it was explained that the cause of spongy hyperostosis could be attributed to tuberculosis, congenital syphilis, or even the pressure caused by carrying water pots on the head (Steinbock, 1976). It was later found that the pathological skulls of children from Chichen Itza, and adults from Pecos were remarkably similar to those of living children with thalassaemia. Both showed marrow hyperplasia and the widening of the diploic space, producing the “hair-on-end” appearance (Steinbock, 1976). It has since been discovered that spongy hyperostosis and the “hair-on-end” pattern can be found in several other hereditary hematological disorders, in addition to thalassaemia and sickle cell disease. In fact, the appearance of the calvarium alone isn’t sufficient for differential diagnosis, since the pattern of bone change is similar for many variations of hematological disorders. Nevertheless, if the “hair-on-end” pattern, or spongy hyperostosis is noted in any skeletal remains, other features should be examined and compared, in order to properly diagnose what type of pathology is exhibited. Figure 15 shows the table of conditions which all produce the “hair-on-end” appearance (Steinbock, 1976:219). 30 Figure 13. (A) Lateral view of step-like depressions in vertebrae. (B) Diagram of the central portion of a normal and collapsed vertebra from ischemia (Milner et. al., 1994: 648). The general pathology associated with sickle cell disease, as well as those homozygous for abnormal hemoglobin C and E, is due to two fundamental processes. The first is that of marrow hyperplasia, which is the process of increased production of red blood cells. The second is that of necrosis of tissues and bone due to the hypoxic state caused by the obstruction of sickled cells. Cortical infarction, or osteonecrosis, leads to new bone formation, cortical thickening, and narrowing of the medullary space. Visibly thickened cortex, the “hair-on-end” appearance in the calvarium, and especially the step-like depressions found in vertebrae may prove useful in examining fossil bones for pathology associated with homozygous hemoglobin S, C, and E, as well as those of thalassaemia. Unfortunately, many of the osteological changes associated with sickle cell disease are also instead the result of other anemias caused by a host of possible conditions. Treponemal infections, various forms of thalassemia, and iron deficiency anemias are among the most common forms of anemia that present pathologies in the skull and long bones. Based on the evidence presented in this thesis, it is 31 Figure 14. X-ray of the “hair-on-end” appearance, caused by widening of the diploic space (Diggs, 1992:157). likely that sickle cell disease existed in early Homo and should be included in the list of possible conditions found in the fossil record. Table 1 lists the skeletal traits of several conditions that should be included in a differential diagnosis of anemia. Pathologies in Homo “Paleopathology is the study of disease of ancient human populations as revealed by their skeletal remains” (Steinbock, 1976:9). In addition, paleopathology is extremely useful in that it can provide information about the antiquity of specific diseases that leave their mark in bones. Many osteological features characteristic of pathologies like sickle cell disease will mimic those of other classified pathologies. The basis of this thesis is to propose the possibility that sickle cell disease evolved prior to both the evolution of Homo sapiens and the origins of agriculture. A brief examination of fossil hominids that may have suffered from sickle cell disease makes a strong case for this argument. 32 Table 1. Skeletal manifestations of selected conditions. Skeletal Traits Thalassemia Frontal and temporal bone thickening X Necrosis of paranasal sinuses, mastoids X "Hair-on-end" pattern of trabeculae X Vertebral compression Osteoporosis of vertebrae X Necrosis and new bone growth in metacarpal and metatarsals X Cortical thickening in the diaphyses of femur, humerus, tibia, fibula X Necrosis of femoral and humeral heads Narrowed medullary cavity of long bones X Tibial bowing Cribra Orbitalia X Sickle cell Anemia Iron Deficiency Anemia X X X X X Treponemal Infection Rickets X X X X X X X X X X X X X X Figure 15. Table of conditions causing “hair-on-end” appearance (Steinbock, 1976: 219). 33 X Two fossil hominids, KNM-WT 15000 and KNM-ER 1808, are strong candidates for this argument, although neither was “diagnosed” as having any form of anemia in their initial analysis. These cases are especially important because the discovery of an intact Homo erectus skeleton is much less common than the discovery of skeletons representing pre-agricultural Homo sapiens, and should be examined carefully for pathology. Skeletal remains exhibiting any type of condition related to abnormal hemoglobin should be examined, even if the cases are of later origin. Of interest to this paper is the report by Hershkovitz and Edelson (1991) on the first identified case of thalassaemia. They state that heterozygotes for thalassaemia major are afforded “a selective advantage over non-carriers in malaria-infested areas because of the malaria parasites’ inability to utilize the affected red cells” (Hershkovitz and Edelson, 1991:49). At one time, it was thought that thalassaemia was present in the very early populations of Sicily in the Upper Paleolithic (Hershkovitz and Edelson, 1991). One analysis of paleodemographic data revealed that the Nea Nikomedeia populations (5500 BCE) were largely affected by malaria, and that their skeletal remains indicate a high frequency of thalassaemia-like conditions Hershkovitz and Edelson, 1991). Enlarged and thickened crania, as well as pathology in the lumbar and thoracic vertebrae have been attributed to thalassaemia (Hershkovitz and Edelson, 1991). The identification of other populations in the Eastern Mediterranean region show that malaria was indeed a major detriment to child and adult health, but that the diagnosis of thalassaemia or sickle cell disease is difficult to make. Most of the skeletal pathology noted in the early part of agricultural society centers around the presence of spongy hyperostosis, also referred to as porotic hyperostosis. “Increase in porotic hyperostosis reflects an increase in some form of anemia, including dietary habits, parasitism and response to infection, as well as to a variety of genetic factors, including sickle cell anemia” (Hershkovitz and Edelson, 1991:50). Regardless of these findings, further research, along with the discovery of more skeletal remains, should serve as tools to pinpoint the specific cause of pathology seem in skeletal remains. What follows is the description of three possible candidates exhibiting pathological features that are consistent with the characteristics of sickle cell disease, all three of which flourished in preagricultural times. 34 Spoor et al. (1998) describe evidence of a calvaria from the Singa region in Sudan which exhibits possible anemia in the temporal bone. Although the Singa calvarium was discovered in 1924, it was neglected due to doubts about it geological age and because it’s abnormal morphology was difficult to interpret. Modern methods, such as oxygen isotope analysis, date the Singa calvaria from 170,000150,000 ky ago, representing a late archaic hominid or an early representative of Homo sapiens (Spoor et al., 1998). It is believed that the cranial vault is pathologically deformed, with the right temporal bone lacking the structures of the bony labyrinth of the inner ear (Spoor et al., 1998). Computed tomography (CT) shows that newly deposited bone destroyed the labyrinth of the inner ear, and, in general, “labyrinthine ossification is consistent with the controversial diagnosis that an anemia causes the characteristic diploic widening of the parietal bosses” (Spoor et al., 1998: 41). Webb (1990) was the first to raise the question of possible pathology, and later suggested that the characteristics of the Singa calvaria are probably related to some form of blood disease. It is stated specifically by Spoor that a common underlying cause of diploic widening and ossification is either a hereditary or acquired blood disorder. Cranial changes are more commonly associated with the thalassaemias than with sickle cell disease, although the latter can not be fully discounted (Serjeant, 1992). Spoor states that Singa does not classify as having servere cranial changes, but that its pattern does mimic that of “sickle cell disease, hereditary spherocytosis and iron deficiency anemia” (Spoor et al., 1998:47). Webb (1990) concludes that the diploic expansion seen in Singa is indicative of hyperplastic marrow, which, as explained earlier, is the main pathogenic process in sickle cell disease (Diggs, 1992; Milner et al., 1994). Spoor concludes by suggesting that the analysis of ancient biomolecules could lead to more definite diagnoses of observed pathologies. The Homo erectus specimen KNM-ER 1808 was found in 1973 from the Upper Member of the Koobi Fora Formation in Kenya (Walker et al., 1982). The claim was that the partial female skeleton showed pathologies consistent with chronic hypervitaminosis A. Hypervitaminosis A is a disorder due to the excessive ingestion of vitamin A, resulting in either acute or chronic forms. Food sources of vitamin A include meats, especially liver, as well as dairy products and carotenoid vegetables. Chronic hypervitaminosis usually occurs for several weeks to a month after ingestion of high levels of vitamin A, resulting in nausea, fever, cracking of the skin, and painful swelling of the appendages. It can 35 also, according to Walker et al., cause “subperiosteal diaphyseal deposits of coarse-woven bone” (1982: 248). Walker et al. (1982) claim that there are sections of the tibial shaft that have given rise to new bone, but are restricted to the outermost cortex. This description also aligns with the pathology consistent with sickle cell disease. However, there is one difference to be noted. Walker et al. (1982) state that the newly formed bone on the outer cortex contains “enlarged, sub-spherical randomly placed lacunae” (Walker, 1982: 248). Figure 16 shows a cross-section at the midshaft of the femur, illustrating the normal bone and the abnormal bone (Walker et. al, 1982: 249). Walker et al. (1982) claim that skeletal manifestations in hypervitaminosis A have been variable. It seems evident from the conclusions of Walker et. al that further research would assist the comparison of bone histology in both sickle cell disease and in hypervitaminosis A. A closer look at others aspects of KNM-ER 1808, such as the diagnostic vertebrae or abnormal metacarpals, may contribute to the case for sickle cell disease. KNM-WT 15000, Homo erectus from northern Kenya, dating to 1.5 million, may also exhibit pathology consistent with sickle cell disease. According to Richard Leakey and Alan Walker (1993), the fossil, known also as the Nariokotome boy, does not show any pathology. They have argued that there are few indications of the cause of death, however, they conclude that the only abnormal feature of the KNM-WT 15000 is a periodontal lesion on the right side of the mandible. This may have been the result of a lethal inflammatory disease, namely septicemia, after the loss of the deciduous second molar (Walker & Leakey, 1993). After examining the literature on Nariokotome boy, it became apparent that there were several features of the skeleton highly characteristic of some type of anemia, possibly inherited. One attribute of sickle cell disease found in KNM-WT 15000 is discussed in reference to language capacity by Walker and Shipman (1996). Walker and Shipman (1996) explain that, except for the skull, the skeleton is very similar to that of modern boys, with just a few small differences. The most striking is that the holes in his vertebrae, through which the spinal cord goes, have only about half the cross-sectional area found in modern humans. One suggested explanation for this is that the boy lacked the fine motor control we have in the thorax to control speech, implying that he did not have the capacity for modern language (Walker and Shipman, 1996). However, if bone growth is inhibited due to the obstruction of the main vertebral artery, the vertebral bodies will become depressed, giving the flattened and widened appearance shown in Figure 13. Studies in vertebral canal width may be helpful in 36 determining if the vertebral canal is narrowed by any form of anemia. If it can be determined that the width of the vertebral canal is narrowed due to hypoxia, then the argument that the narrow width is due to the incapacity for language must be reexamined. This can de bone with a combined histological analysis of the vertebra itself and differential Figure 16. Cross section at midshaft of femur. a and b show normal bone, while d is typical of the abnormal bone. c is a junction point where normal (a,b) meet abnormal bone (d) (Walker et al., 1982: 249). diagnosis of other possible bones pathologically affected by some form of anemia. In addition to the vertebrae, there is also evidence of pathology in the Nariokotome femurs (Walker & Leakey, 1993). Both genetic and acquired forms of anemia affect the femoral and humeral head. The femoral neck of Nariokotome is described as having particularly strong vascular striae running from the superomedial surface to the lesser trochanter as well as an irregular pit in the region of the insertion of the gluteus minimus (1993: 144). In addition, the region of the metaphysis just above the patellar groove is marked by large vascular foramina. Walker and Leakey do not imply that these are abnormal features. However, the most interesting aspect in the description of the femurs is that both have “pronounced subtrochanteric platymeria, a very thick shaft, and a narrow medullary cavity” (Walker & Leakey, 1993: 145). This feature is seen in cases of sickle cell anemia, as well as various forms of thalassemia. Nevertheless, the description of Nariokotome alone warrants a further look at the possibility of pathology, including either inherited or acquired forms of anemia. One particular case, presented by Hershkovitz and Edelson (1991) on the New Nikomedeia populations may shed light on the case of KNM-WT 15000. A perfectly intact humerus of a 16 yearold male, named Homo 25, was found which exhibited several similar pathologic features to that of KNM-WT 15000. The radiographs showed that the humerus of Homo 25 had marked osteoporosis, as well as areas of local infarction and thickened cortex. Both KNM-WT 15000 and Homo 25 are 37 described as exhibiting the same characteristic features, which typify both sickle cell disease and thalassaemia. Thickened shafts of the long bones and narrowed medullary cavities are the results of pathologies in both sickle cell disease and other types of inherited anemia, specifically of marrow hyperplasia and osteonecrosis. Of less significance is the description of the upper left tibia of WT-KNM 15000. Walker and Leakey explain that there are a “peculiar” series of grooves that cut across the surface of the bone as a result of root erosion. Their analysis is by far the most extensive done on the Nariokotome skeleton, however, this description is still vague and should be examined further. Furthermore, since Nariokotome is one of the most complete skeletons ever found, there should be a sufficient amount of work done on all the existing bones, beyond description of orientation and measurement, including into areas of possible pathology. If sickle cell disease, (and other related hemoglobin disorders conferring resistance to malaria infection), originated prior to agriculture is to be tested, the pathologies associated with the disease should be included in times where a differential diagnosis is necessary. Infectious diseases, particularly the various treponemal infections, may mimic those of thalassaemia and sickle cell disease. Since treponemal infection causes bone lesions similar to sickle cell disease, it can be difficult to distinguish which agent caused the lesions, especially when “the specimen consists of a solitary bone or bone fragment because the rest of the skeleton had deteriorated or was left behind as having no scientific value” (Steinbock, 1976: 94). Even when the entire skeleton is available, the similarities between sickle cell disease and yaws, a type of treponemal infection, exist to a great enough extent that they should both be considered when pathology is found. Dactylitis, or hand-foot syndrome, is one such pathology that has been described in context with both sickle cell disease and treponemal infection. The results of this thesis make a case for including sickle cell disease in a list of possible pathologies in the fossil record as early as Homo erectus. The extraction of DNA from the fossil record in the future would more solidly confirm the presence of sickle cell disease in early Homo. Until then, it is necessary to closely examine the diagnostic features of sickle cell disease in relation to other pathologies, considering a diagnosis of sickle cell disease as one possibility. 38 CHAPTER 5 CONCLUSIONS The evidence provided throughout this thesis presents a strong argument for sickle cell disease and associated homozygous conditions originating significantly earlier than currently thought. It has been shown that the historical evidence of malaria alone contributes only a fragment to our knowledge of the endemic disease. The Livingstone hypothesis (1958) states that the sickle cell trait originated as the result of slash-and-burn agriculture, which increased the rate of malaria, and thus created pressure on the gene pool to select the trait. Supporters of the Livingstone hypothesis believe that the most common form of Plasmodium, P. falciparum, is a young species and is the result of a recent host switch from a form of avian Plasmodium. However, new studies in molecular biology and gene sequencing, conducted by Escalante and Ayala (1994, 1996) have shown that the most virulent Plasmodium parasites infective to humans are as old, or older, than the divergence of human and chimpanzee from their most common ancestor. The modernization of genetic sequencing techniques proves to be extremely definitive in such matters as the evolutionary history of a species. However, even with the case of the genus Plasmodium, the age of origin is derived differently depending on which specific gene sequences are analyzed. Hopefully, further molecular studies will combine various methods and techniques to improve the reliability and accuracy of the results. This paper adheres to the conclusions of Escalante and Ayala (1996), which derives an age of at least 8-10 mya for the origin of Plasmodium parasites infective to humans and chimpanzees. The analysis of small subunits of ribosomal RNA combined with that of the circumsporozoite protein (CSP) provides a thorough examination of the genetic makeup of the eleven Plasmodium species investigated by Escalante and Ayala. As shown in Chapter 2, P. falciparum aligns with P. reichenowi, the chimpanzee parasite, with a bootstrap value of 100%. This strongly supports the claim that Plasmodium parasites infective to both man and chimpanzee originated and co-existed at a similar time. Chapter 3 examined the various genetic defense mechanisms that have been selected for over time as balanced polymorphisms. There are hundreds of hemoglobin mutations, most of which have occurred as the result of single point mutations. This implies that hemoglobin mutations have been 39 occurring for thousands of years, and that it has taken a significant amount of time to accumulate several varieties of abnormal hemoglobin, that, when heterozygous, confer natural resistance to malarial infection. Hopefully, future molecular studies will delve more deeply into the estimated timeframes during which abnormal hemoglobins, including type S, C, E, and those of the thalassaemia, were enjoying positive selection. Once it can be established that the selection for the sickle cell trait (HbS) occurred prior to the evolution of Homo sapiens, it follows that the frequency of homozygosity for the trait would begin to rise. Once there is sufficient distribution of individuals heterozygous for the sickle cell trait, there will be a steady rise in the number of individuals that inherit two copies of the trait. Although it should be noted that many individuals homozygous for the sickle cell trait (HbS) will not live into adulthood, the work of Serjeant (1992) and Sharufa (1982) document both younger individuals with benign cases of sickle cell disease as well as a healthy number of survivors over the age of thirty. As discussed in Chapter 4, the skeletal pathology of sickle cell disease is distinct at times, vague in others. Spongy hyperostosis, a pathology frequently found in the fossil record, can be the result of sickle cell disease, thalassaemia, any many times, non-hereditary forms of anemia. The fact that many different forms of anemia leave the same signature, or host of features in pathological bone, makes it difficult to pinpoint the exact cause of disease. However, evidence provided by modern clinical research serves as an important tool for use in differential diagnosis. Histological analysis may be extremely useful in the diagnosis. Cellular structure can be indicative of heredity blood disorders, so histological analysis of skeletal material should be considered as a diagnostic tool. Macroscopic examination of bone lesions may indicate a variety of causes such as rickets, inflammation cause by osteomyelitis or both acquired and inherited forms of anemia (Bianco and Ascenzi, 1993). However, light microscopy can show that there are slight differences at the microscopic level. For example, in the case of sickle cell anemia, the internal lamina is not involved, while the external lamina is almost completely destroyed (Bianco and Ascenzi, 1993). In the case of thalassemia and sickle cell disease, the trabeculae of the diploe take on a parallel orientation on the external surface, whereas in rickets, “very small squamous appositions cover the external lamina and give the bone a porotic aspect” (Bianco and Ascenzi, 1993: 187). Furthermore, the use of ancient DNA (aDNA) may be helpful in tracing pathology in the fossil record. Although there are multiple disadvantages in the study, extraction and reliability of ancient DNA, 40 it is possible to test for inherited disease using polymerase chain reaction tests. In the future, when pathological skeletal material is recovered from the fossil record, every attempt should be made to differentially diagnosis types of anemia. It is suggested that the traits should first be examined in relation to other diseases that leave a similar skeletal signature. If there is a likelihood of the presence of sickle cell anemia, further studies should be considered. If it can be established that sickle cell disease is of much older origin than currently thought, it would be necessary to reexamine all other hypotheses regarding the first cases of malaria, as well as the evolution of the sickle cell trait and associated hemoglobin abnormalities. These conclusions are essential in order to reshape current knowledge of the relationship between malaria and sickle cell disease. As the search continues for more skeletal material of any age, special efforts should be made to identify all forms of pathology. By no means should sickle cell disease, or any other type of inherited hemoglobin disorder, be discounted from the list of possible pathological conditions on the basis of age alone. The assumption that sickle cell disease did not exist prior to agriculture seriously hinders the field of paleopathology, and, based on the evidence presented in this thesis, should be reexamined. 41 BIBLIOGRAPHY Bohrer, SP (1970) Acute long bone diaphyseal infarcts in sickle cell disease. Br. J. Radiol. 43: 685-97. Bianco, P, Ascenzi, A (1993) Palaeohistology of human bone remains: A critical evaluation and an example of its use. In Histology of Acient Human Bone: Methods and Diagnosis, Grupe, Garland, eds. 185-201. London: Springer-Verlag. Bray, RS (1969) Malaria in Chimpanzees. The Chimpanzee 1:413-424. Brogden, B, Lecklitner, M, Machen, B, Williams, J (1992) Radiological Implications of Sickle Cell Disease. In Pathophysiology, Diagnosis and Management, Mankadd & Moore, Eds. 321351. Connecticut: Prager Publishers. Brown, F, Harris, J, Leakey, R and Walker, A (1985) Early Homo erectus skeleton from west Lake Turkana, Kenya. Nature 316:788-792. Desowitz, R (1991) The Malaria Capers. New York: Norton & Company. Diggs, L (1992) Anatomical Lesions in Sickle Cell Hemoglobinopathies. In Sickle Cell Disease: Patho-Physiology, Diagnosis and Management, Mankadd & Moore, eds. 142-200. Connecticut: Prager Publishers. Eaton, J (1994) Malaria and the Selection of the Sickle Gene. In Sickle Cell Disease Basic Principles And Clinical Practice, Embury, S, Hebbel, R, eds. 13-18. Embury, Stephen, Hebbel, R., Mohandas, N., Steinberg, M. (1994) Sickle Cell Disease: Basic Principles and Clinical Practice. New York: Raven Press. Escalante A, Ayala, F (1996) Molecular Paleogenetics: The evolutionary history of Plasmodium and related Protists. In Evolutionary Paleogenetics. D. Jablonski, D. H. Erwin, eds. 21-41. Chicago: The University of Chicago Press. Escalante, A. E. Barrio, F Ayala (1995) Evolutionary origin of human and primate malarias: Evidence from the circumsporozoite protein gene. Molecular Biology and Evolution 12: 616-626. Escalante, A, Ayala, F (1994) Phylogeny of the malarial genus Plasmoium derived from rRNA gene Sequences. Proceedings of the National Academy of Sciences 91: 11373-77. Garnham, P.C. (1966) Malaria parasites and other haemosporidia. Oxford: Blackwell Scientific. 42 Gilles, H.M. (1999) The Malaria Parasites. In Gilles, HM, Warrell Bruce-Chwatts Essential Malarialogy, 3rd edition, London: Edward Arnold. Harder, B (2001) The Seeds of Malaria: Recent Evolution cultivated a deadly scourge. Science 160 (19): 296. Hershkovitz, I, Edelson, G. (1991) The first identified case of thalassemia? Human Evolution 6 (1): 49-54. Hershkovitz, I, Rothschild, B, Latimer, B, Dutour, O, Leonetti, G, Greenwald, C, Rothschild, C and Jellema, L (1997) Recognition of Sickle Cell Anemia in Skeletal Remains of Children. Am. J. Phys. Anth., 104:213-226. Hill, A, Weatherall, D (1998) Host Genetic Factors in Resistance to Malaria. In In Malaria: Parasite Biology, Pathogenesis, and Protection. Sherman, Irwin, ed. 11-25. Washington: Library of Congress. Hoeprich, P. (1989) Host-parasite relationships and the pathogenesis of infectious disease. In Infectious Diseases, ed. P. Hoeprich, M. Jordan, 41-53. Philadelphia: J.B. Lippincott. Hommel (1999) Biology of the parasite and host-parasite relationships. In Protozoal Diseases. H. Gilles, 25-74. New York: Oxford University Press. Kazazian, H (1990) The thalassaemia syndromes: molecular basis and prenatal diagnosis in 1990. Semin. Hematol. 27 209-28. Knell, A (1991) Malaria. New York: Oxford University Press. Kurnit, D (1979) Evolution of sickle variant gene. In Sickle Cell Disease. Serjeant, ed. 83-96. New York: Oxford University Press. Lehman, H (1954) Origin of the sickle cell. In Sickle Cell Disease. Serjeant, ed. 16-28. New York: Oxford University Press. Lopez-Antunano, F Schumunis, G. (1993) Plasmodia of humans. In Parasitic Protozoa, 2d ed., vol. 5, ed. J. Kreier, 135-265. New York: Academic Press. Livingstone, F (1958) Anthropological implications of sickle cell gene distribution in West Africa. Am Anth. 60: 533-562. Livingstone, F (1971) Malaria and human polymorphisms. Ann Rev of Genetics 5:33-64. 43 Mankadd, V, Hoff, C (1992) Epidemiology and Population Genetics of Sickle Cell Disease and the Sickle Cell Trait. In Sickle Cell Disease: Pathophysiology, Diagnosis and Management, Mankadd & Moore, Eds. 1-14. Connecticut: Prager Publishers. Mays, S (1998) The Archaeology of Human Bones. New York: Routledge Press. Milner, P, Joe, C, Burke, G (1994) Bone and Joint Disease. In Sickle Cell Disease: Basic Principles And Clinical Practice, Embury, S, Hebbel, R, eds. 13-18. Moore, R (1992) Pathophysiology of the Sickle Cell. In Sickle Cell Disease: Pathophysiology, Diagnosis and Management, Mankadd & Moore, eds. 142-200. Connecticut: Prager Publishers. Nagel, R (1994) Origins and Dispersion of the Sickle Gene. In Sickle Cell Disease: Basic Principles and Clinical Practice. New York: Raven Press. Rachmilewitz, E, Levi, S, Huisman, T (1974) High frequency of hemoglobin C in an Israel Bedouin tribe. In Sickle Cell Disease. Serjeant, G, ed. New York: Oxford University Press. Serjeant, G (1992) Sickle Cell Disease. New York: Oxford University Press. Sherman, Irwin (1998) A Brief History of Malaria and Discover of the Parasite’s Life Cycle. In Malaria: Parasite Biology, Pathogenesis, and Protection. Sherman, Irwin, ed. 11-25. Washington: Library of Congress. Shurafa, M, Prasad, A, Rucknagel, D and Kan, Y (1982) Long survival in sickle cell anemia. Am. J. Hematol. 12: 357-65. Sinden, R. (1998) Gametocytes and Sexual Development. In Malaria: Parasite Biology, Pathogenesis, and Protection. Sherman, Irwin, ed. 11-25. Washington: Library of Congress. Spoor, F, Stringer, C, Zonneveld, F (1998) Rare Temporal Bone Pathology of the Singa Calvaria from Sudan. Am. J. Phys. Anth. 107: 41-50 Steinbock, Ted (1976) Paleopathological Diagnosis and Interpretation. Springfield: Bannerstone House. Stuart-Macadam, P (1989) Nutritional Deficiency Diseases: A Survey of Scurvy, Rickets, and IronDeficiency Anemia. In Reconstruction of Life from the Skeleton. Yasar Iscan, M, Kennedy, K, eds. 201-222. New York: Alan R. Liss. Stuart-Macadam, P, Kent, S (1992) Diet, Demography, and Disease: Changing Perspectives on Anemia. New York: Aldine de Gruyter. 44 Trigg, P, Kondrachine, A (1998) The Current Global Malaria Situation. In Malaria: Parasite Biology, Pathogenesis, and Protection. Sherman, Irwin, ed. 11-25. Washington: Library of Congress. Walker, A, and Leakey, R (1993) The Nariokotome Homo Erectus Skeleton. Cambridge: Harvard University Press. Walker, A, and Shipman, P (1996) Wisdom of the Bones. New York: Alfred A. Knopf. Walker, A, Zimmerman, MR and Leakey, R (1982) A possible case of hypervitaminosis A in Homo erectus. Nature 296:248-250. Waters, A, Higgins, D., McCutchan, T. (1991) Plasmodium falciparum appears to have arisen as a result of lateral transfer between avian and human hosts. Proceedings of the National Academy of Sciences 88: 3140-44. Webb, S (1990) Cranial Thickening in an Australian hominid as a possible palaeoepidemiological indicator. Am. J. Phys. Anth. 85:403-411. Wilairatana, P., Looareesuwan S., Gilles, H. (1994) Pathogenesis, pathology, clinical manifestations and differential diagnosis. In Protozoal Diseases. H. Gilles., ed. 25-74. New York: Oxford University Press. 45 BIOGRAPHICAL SKETCH Kellei Latham Jefferson was born February 25, 1976 in Huntsville, Alabama. She attended the Universities of Alabama in Birmingham, Tuscaloosa, and Huntsvile. Shen then went on to graduate from the Ohio State University in 1999, earning a bachelor of science degree in International Relations, with a focus on peace and conflict resolution. She then entered the graduate program at Florida State University in Anthropology, with an interest in bone pathology and forensics. 46