Download The Possible Selection of the Sickle Cell Trait in Early

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