Ancestry and Pathology in King Tutankhamun's Family FREE

The 18th dynasty (circa 1550-1295 BC) of the New Kingdom (circa 1550-1070 BC) was one of the most powerful royal houses of ancient Egypt. The pharaoh Akhenaten, who ruled from circa 1351 to 1334 BC, is considered one of the most controversial of the Egyptian pharaohs, because his attempt to radically transform traditional religion affected all facets of society and caused great turmoil.

Akhenaten's eventual successor, Tutankhamun, is probably the most famous of all pharaohs, although his tenure was brief. He died in the ninth year of his reign, circa 1324 BC, at age 19 years. Little was known of Tutankhamun and his ancestry prior to Howard Carter's discovery of his intact tomb (KV62) in the Valley of the Kings in 1922, but his mummy and the priceless treasures buried with him, along with other important archeological discoveries of the 20th century, have provided significant information about the boy pharaoh's life and family.

Because Tutankhamun died so young and left no heirs, numerous speculations on familial disease have been made. The presence of disease is further supported by numerous reliefs, statuettes, and other sculptures of Akhenaten and his family dating from the Amarna period (circa 1353-1323 BC). These artifacts show the royalty of that era as having a somewhat androgynous appearance or a bizarre form of gynecomastia. Specific diseases that have been suggested to explain this appearance include Marfan syndrome, Wilson-Turner X-linked mental retardation syndrome, Fröhlich syndrome (adiposogenital dystrophy), Klinefelter syndrome, androgen insensitivity syndrome, aromatase excess syndrome in conjunction with sagittal craniosynostosis syndrome, or Antley-Bixler syndrome or a variant form of that syndrome.^1- 4 However, most of the disease diagnoses are hypotheses derived by observing and interpreting artifacts and not by evaluating the mummified remains of royal individuals apart from these artifacts.

To shed light on the putative diseases and causes of death in Tutankhamun's immediate lineage, we first used molecular genetic methods to determine kinship within that lineage. Whereas some individual relationships were known from historical records, the identity of most of the mummies under investigation was still uncertain. We also searched specifically for pathologies, inherited diseases, and causes of death. For example, many scholars have hypothesized that Tutankhamun's death was attributable to an accident, such as a fall from his chariot or a kick by a horse or other animal; septicemia or fat embolism secondary to a femur fracture; murder by a blow to the back of the head; or poisoning.^5- 10 We had access to mummies that had never before been studied with the methods we used.

In addition to Tutankhamun, 10 mummies possibly or definitely closely related in some way to Tutankhamun were chosen for this 2-year project; of these, the identities were certain for only 3. In addition to these 11 mummies, 5 other royal individuals dating to the early New Kingdom were selected that were distinct from the putative members of the Tutankhamun lineage. These 5 mummies were used as a morphological (excluding Ahmose-Nefertari) and genetic (excluding Thutmose II) control group. All mummies are listed in Table 1, and full-body computed tomography reconstructions of the mummies are available here.

All of the mummies, except for that of Ahmose-Nefertari, were scanned using a multidetector computed tomography unit (Somatom Emotion 6; Siemens Medical Solutions, Malvern, Pennsylvania) installed on a truck. The tomography unit was used to examine the mummy of Tutankhamun and those of the 2 women from tomb KV35 in Luxor as well as the rest of the mummies at the Egyptian Museum in Cairo (eAppendix). Cephalic indices of mummy heads were determined according to the method of Weber et al.¹¹

We adopted the previously published criteria for ancient DNA authentication, which form a consensus outline for executing research studies using ancient DNA (eAppendix).^12- 13 Sampling of bone tissue and DNA extraction and purification were performed according to protocols previously published.^14- 15 Negative and blank extraction controls were processed along with each sample. In addition, water and other aqueous polymerase chain reaction (PCR) components were monitored using the sensitive internal-Alu-PCR protocol¹⁶ to assess contamination with modern human DNA.

Sixteen Y-chromosomal short tandem repeats (DYS456, DYS389I, DYS390, DYS389II, DYS458, DYS19, DYS385, DYS393, DYS391, DYS439, DYS635, DYS392, Y-GATA-H4, DYS437, DYS438, DYS448) were amplified according to the manufacturer's protocol using the AmpF\STR Yfiler PCR amplification kit (Applied Biosystems, Foster City, California). The Identifiler kit and the AmpF\STR Minifiler kit (Applied Biosystems) were used for amplification of 8 polymorphic microsatellites of the nuclear genome (D13S317, D7S820, D2S1338, D21S11, D16S539, D18S51, CSF1PO, FGA).

To test for Plasmodium falciparum DNA, PCR primers were designed that specifically amplify small subtelomeric variable open reading frame (STEVOR), apical membrane antigen 1 (AMA1), and merozoite surface protein 1 (MSP1) gene fragments with sizes of 100 to 250 base pairs (bp). PCR products and cloned DNA fragments were sequenced by the Sanger method (eAppendix). Purified amplicons were run on a genetic analyzer (ABI Prism 3130, Applied Biosystems). Microsatellites were interpreted with Data Collection Software version 3.0 and GeneMapper ID version 3.2 (Applied Biosystems). Lasergene version 8.0 (DNAstar, Madison, Wisconsin) and BioEdit version 7.0.9 (Ibis Biosciences, Carlsbad, California) were used to establish multisequence alignments (eAppendix).

To elucidate the genealogy in Tutankhamun's family, microsatellite markers were used to achieve genetic fingerprints of all mummies. All 8 females tested were negative for the examined polymorphic Y-chromosomal loci, underlining the specificity of the approach. The repeated search for hemizygous Y alleles in the males yielded few results, with differing success in the various markers contained in the multiplex PCR kit used. Markers DYS393 and Y-GATA-H4 showed identical allele constellations (repeat motif located in the microsatellite allele reiterated 13 and 11 times, respectively) in Amenhotep III, KV55, and Tutankhamun but different allelotypes in the nonrelated CCG61065 sample from TT320 (9 and 9, respectively). Syngeneic Y-chromosomal DNA in the 3 former mummies indicates that they share the same paternal lineage.

These results were repeatedly obtained with DNA extracted from 2 to 4 different biopsies per mummy; moreover, they differed from the Y profiles of the male laboratory staff and were independently reproduced twice in a second laboratory physically isolated from the first, data-generating laboratory.

An up to 30-fold testing of polymorphic autosomal microsatellite loci via the combined use of the Identifiler and AmpF\STR Minifiler kits (Applied Biosystems) yielded complete data sets for all 8 markers in 7 mummies (Thuya, Yuya, Amenhotep III, Tutankhamun, KV55, and both female mummies from KV35) but only partial data for both KV62 fetuses and the KV21A and KV21B mummies (Figure 1). Repeated attempts to complete the profiles in the 4 latter mummies were not successful; however, we were able to replicate some of the results for the previous mummies more than 4 times in the second, independent laboratory (Figure 1). Moreover, because these profiles differed from those of the laboratory staff and were not identical to the ones established for the control group, the data were considered authentic.

Based on the partial Y-chromosomal information on the amount of autosomal half-allele sharing and family trio likelihood calculation, the most plausible 5-generation pedigree was constructed. We identified Yuya and Thuya as great-grandparents of Tutankhamun, Amenhotep III and KV35EL as his grandparents, and the KV55 male and KV35YL as his sibling parents (Figure 1, Figure 2, and online interactive kinship analysis and pedigree; for details on kinship statistics, see eAppendix).

The most prominent feature exhibited by the art of the pharaoh Akhenaten, seen also to a lesser degree in the statues and reliefs of Tutankhamun, is a markedly feminized appearance (eFigure 1A-C), reasonably suggesting some form of gynecomastia or Marfan syndrome as an underlying disease.^1- 4 However, putative breasts in Tutankhamun and his father Akhenaten (KV55) cannot be determined, because KV55 is a mummified skeleton and Tutankhamun lacks the frontal part of the chest wall. The penis of Tutankhamun, which is no longer attached to the body, is well developed. Furthermore, the pelvic bones of Tutankhamun are almost entirely missing, and the pelvis of KV55, which is present but fragmented, does not show feminine traits after reconstruction using computed tomography (eAppendix, eFigure 1D-G, and online interactive feature).

One of the obvious features of Marfan syndrome is dolichocephaly.^17- 19 With the exception of Yuya (cephalic index, 70.3), none of the mummies of the Tutankhamun lineage has a cephalic index of 75 or less (ie, indicating dolichocephaly). Instead, Akhenaten has an index of 81.0 and Tutankhamun an index of 83.9, indicating brachycephaly. From the control group, Thutmose II and the TT320-CCG61065 mummy show dolichocephaly, with cephalic indices of 73.4 and 74.3, respectively. Because there is no sign of premature closure of sutures, none of the skull shapes can be considered pathological. The complex diagnosis of Marfan syndrome is based on certain combinations of major and minor clinical features.¹⁸ Following this classification, a Marfan diagnosis cannot be supported in these mummies (Table 2). Antley-Bixler syndrome is also excluded in Tutankhamun and Akhenaten because their brachycephaly is not attributable to craniosynostoses, and further signs of Antley-Bixler or other syndromes are missing or unspecific.

Tutankhamun's mummy was examined several times radiologically.^20- 23 Our inspection of the skull and trunk did not reveal novel information, but detailed examination of the king's feet yielded new data. Compared with the normal anatomy of the foot (Figure 3), the right foot had a low arch (Rocher angle, 132°; normal value, 126°). The medial longitudinal arch of the left foot was slightly higher than normal (Rocher angle, 120°) (Figure 4A), with the forefoot in supine and inwardly rotated position akin to an equinovarus foot deformity (Figure 4B). There were no pathological findings on the bone structure of the right metatarsal heads (Figure 5A). In contrast, the left second metatarsal head was strongly deformed and displayed a distinctly altered structure, with areas of increased and decreased bone density indicating bone necrosis (Figure 5B). The study further showed a widening of the second metatarsophalangeal joint space, with a normal articulating surface of the proximal phalanx. The third metatarsal head was only slighty deformed; the bony structure, however, showed signs of bone necrosis. The remaining left metatarsal heads appeared to be of normal structure (Figure 5B). The plantar surface of the left second metatarsal head shows a crater-shaped bone and a soft tissue defect in the area of bone necrosis (Figure 5C). The second and third toes on the left foot are in abduction. The second toe is shortened because it lacks the middle phalanx (oligodactyly [hypophalangism]). The proximal phalanx directly articulates with the distal phalanx (Figure 5D).

Except for Ahmose-Nefertari, all remaining mummies were subjected to radiological analyses. Along with various bony malformations (eg, cleft palate, kyphoscoliosis, clubfeet, flat feet) in the remaining mummies, indications of bone degeneration, neoplastic changes, and trauma were also found. These various findings are listed in Table 3 and are described in the eAppendix.

Various infectious diseases are suspected or known to have been prevalent in antiquity,^24- 27 and some are described in remarkable detail in Egyptian papyri (eg, Papyrus Ebers, circa 1520 BC). Positive results were not found for pandemic plague (Black Death, bubonic plague), tuberculosis, leprosy, or leishmaniasis, but we identified DNA of P falciparum (the malaria parasite) in several of the royal mummies. Amplification of the P falciparum STEVOR gene family²⁸ repeatedly yielded 149-bp and 189-bp amplicons for Tutankhamun and the TT320-CCG61065 mummy and also yielded a faint PCR band using DNA of the Yuya mummy. This result was replicated in further PCRs using DNA from other biopsies (for details on STEVOR data see eAppendix and eFigure 2).

To consolidate or disprove this result, we targeted a further Plasmodium gene using new DNA extracts from the royal mummies in our study. We identified 4 mummies as positive for AMA1, a merozoite protein responsible for the successful binding of the parasite to the erythrocyte membrane, by amplifying DNA fragments locating to the conserved region of the AMA1 gene (Figure 6). The AMA1 PCR fragments were obtained for all mummies testing positive in the earlier STEVOR assays (ie, Tutankhamun, Yuya, TT320-CCG61065). In addition, we also obtained a positive typing for Thuya. Repetition of these experiments in the second laboratory using DNA extractions from new biopsies confirmed the previous data (Figure 6; for details on AMA1 data, see eAppendix).

In addition to the STEVOR and AMA1 genes, we attempted amplification of alleles of the MSP1 and MSP2 genes specific to P falciparum. Because of the fragmented nature of the ancient DNA, we did not obtain positive amplifications when targeting the larger (>400 bp) PCR alleles of the MSP2 gene but were successful in amplifying different alleles of the MSP1 gene (for details on MSP1 data, see eAppendix).^29- 30 Using extracts from Tutankhamun and Yuya, we repeatedly amplified the RO33 and MAD20 alleles, which is indicative of at least a double infection with the P falciparum parasite. The DNA of Thuya yielded amplicons for the RO33 allele. The DNA of TT320-CCG61065 was refractory to MSP1 amplifications. Cloning the obtained allelic fragments into TA plasmid vectors and subsequent Sanger sequencing of 21 clones designated the sequences as specific for MSP1 (eAppendix).

More than 55 bone biopsies were used to elucidate the individual relationships of 18th-dynasty individuals, with the result that several of the anonymous mummies or those with suspected identities are now able to be addressed by name. These include KV35EL, who is Tiye, mother of Akhenaten and grandmother of Tutankhamun, and the KV55 mummy, who is most probably Akhenaten, father of Tutankhamun (Figure 2, eAppendix, and online interactive kinship analysis and pedigree). The latter kinship is supported in that several unique anthropological features are shared by the 2 mummies and that the blood group of both individuals is identical.^31- 32

Macroscopic and radiological inspection of the mummies did not show specific signs of gynecomastia, craniosynostoses, Antley-Bixler syndrome or deficiency in cytochrome P450 oxidoreductase, Marfan syndrome, or related disorders (eAppendix, Table 2). Therefore, the particular artistic presentation of persons in the Amarna period is confirmed as a royally decreed style most probably related to the religious reforms of Akhenaten. It is unlikely that either Tutankhamun or Akhenaten actually displayed a significantly bizarre or feminine physique.

It is important to note that ancient Egyptian kings typically had themselves and their families represented in an idealized fashion. A recent radiographic examination of the Nefertiti bust in the Berlin Museum illustrates this clearly by showing that the original face of Nefertiti, present as a thin layer beneath the outer surface, is less beautiful than that represented by the artifact.³³ Differences include the angles of the eyelids, creases around the corners of the mouth on the limestone surface, and a slight bump on the ridge of the nose.³⁴ Thus, especially in the absence of morphological justification, Akhenaten's choice of a “grotesque” style becomes even more significant.

Tutankhamun had a juvenile aseptic bone necrosis of the left second and third metatarsals (Köhler disease II, Freiberg-Köhler syndrome). The widening of the metatarsal-phalangeal joint space, as well as secondary changes of the second and third metatarsal heads, indicate that the disease was still flourishing at the time of death.³⁵ Bone and soft tissue loss at the second metatarsal phalangeal articulation could further indicate that an acute inflammatory condition was present on the basis of an ulcerative osteoarthritis and osteomyelitis. The congenital equinovarus deformity (pes equinovarus) together with the malformed second toe of the left foot (oligodactyly [hypophalangism]) transferred additional joint load to the right foot, causing flattening of the foot arch (pes planus).

There is evidence that Tutankhamun may have had this impairment for quite some time. The walking disability can be substantially aided by the use of a cane. Howard Carter discovered 130 whole and partial examples of sticks and staves (eFigure 3A) in the king's tomb, supporting the hypothesis of a walking impairment.³⁶ Traces of wear can be seen on a number of the sticks, demonstrating that they were used in the king's lifetime (eFigure 3B). Additional evidence for some sort of physical disability is found in a number of 2-dimensional images from Tutankhamun's reign that show him seated while engaged in activities for which he normally should have been standing, such as hunting (eAppendix and eFigure 3C).^37- 38

Macroscopic studies revealed areas of patchy skin changes on the pharaoh's left cheek and neck of uncertain anamnesis, possibly indicating an Aleppo boil, a plague spot, an inflamed mosquito bite, or a mummification artifact.³⁹ However, the genetic identification and typing of plasmodial DNA in Tutankhamun, Thuya, Yuya, and TT320-CCG61065 showed that they must have had malaria tropica, the most severe form of malaria (eAppendix).

Literary evidence for malaria infection dates back to the early Greek period, when Hippocrates described the periodic fever typical of this disease.⁴⁰ Although it is believed that malaria widely affected early populations before Hippocrates,^27,41 until now only 1 report using immunological tools⁴² and few molecular genetic studies have clearly identified P falciparum in ancient specimens.^43- 46 We not only identified this parasite in our sample but also observed individual differences in some of the gene sequences as well as different MSP1 allele constellations in the 4 positive mummies. The diversity of plasmodial DNA (ie, variability in the genes' base order, length polymorphisms, or both) is a well-known phenomenon; however, some of the base deviations were not found in current DNA databases. Further research is required to typify these alterations in more detail and to assign these potentially unknown patterns to ancient Egyptian Plasmodium strains that date back to 3300 to 3400 years before present.

To our knowledge, this is the oldest genetic proof for malaria in precisely dated mummies. When the infection occurred, its severity, and whether it could have caused the death in the 4 mummies testing positive is not known. Preliminary data show that Tutankhamun and Yuya had multiple infections, as could be seen by the presence of the 2 P falciparum alleles MAD20 and RO33 of the MSP1 in the extracts. In contrast, and taking only the MSP1 test system into account, Thuya was infected by only 1 strain, which displayed the RO33 allele.

To date, no association has been found between P falciparum MSP1 genotypes and the clinical status of persons affected.⁴⁷ We note that mixed P falciparum infections were detected in up to 78% of a contemporary sampling, and even isolates from symptomatic children contained more than 1 Plasmodium clone.^47- 48 Thus, multiple infections appear to be the norm rather than the exception. Moreover, the MSP1 allele frequencies tend to vary largely in different, sometimes even neighboring, areas but also over time.²⁹ Thus, the prevalence rate of infection is not known—nor is it known if malaria was an epidemic or an endemic disease and how widely it was distributed in ancient Egypt.

Unfortunately, there is also no distinct evidence in ancient Egyptian texts of treatments for malaria, and there are no references to the fevers and chills associated with the disease.⁴⁹ However, the Nile Delta and the fringes of the Nile Valley were marshy areas and thus excellent breeding grounds for the mosquito genus Anopheles. Interestingly, mosquitoes are mentioned in at least 1 ancient text,⁵⁰ and it has also been suggested that the wooden frame of Queen Hetepheres (fourth dynasty) served as the support for a mosquito net.⁵⁰ Herodotus also mentions that Lower Egypt was infested with mosquitoes or other insects and that people slept under nets to avoid them.⁵¹ Since there is nothing in the historical or archeological record that speaks against the widespread presence of this carrier in Pharaonic times, there is no evidence that can be used to argue against the diagnosis of malaria.

Caution must be taken when interpreting cause of death in these mummies. It can be speculated that Yuya and Thuya had malaria, but it is not known if this was lethal (Table 3). Surprisingly, both individuals had reached an advanced (for the time) age of approximately 50 years or older (Table 1). This means either that the infection took place quite late in their lifetime, that they enjoyed strong genetic fitness, or that they aquired a partial immunity against the pathogen during their lives. Not every person infected with P falciparum becomes gravely ill, and this is especially true in populations that have been exposed to malaria pathogens over long periods.⁵² If Yuya and Thuya spent much of their time living in malaria-endemic areas close to the marshes of the Nile River, partial immunization may have contributed to their survival.

On the other hand, Tutankhamun had multiple disorders, and some of them might have reached the cumulative character of an inflammatory, immune-suppressive—and thus weakening—syndrome (Table 3). He might be envisioned as a young but frail king who needed canes to walk because of the bone-necrotic and sometimes painful Köhler disease II, plus oligodactyly (hypophalangism) in the right foot and clubfoot on the left. A sudden leg fracture²³ possibly introduced by a fall might have resulted in a life-threatening condition when a malaria infection occurred. Seeds, fruits, and leaves found in the tomb, and possibly used as medical treatment, support this diagnosis (eAppendix, eFigures 3D and 3E).^{24- 25,53- 57}

In conclusion, this study suggests a new approach to research into the molecular genealogy and pathogen paleogenomics of the Pharaonic era. With additional data, a scientific discipline called molecular Egyptology might be established and consolidated, thereby merging natural sciences, life sciences, cultural sciences, humanities, medicine, and other fields.