Ancestry and Pathology in King Tutankhamun’s Family. JAMA, February 17, 2010—Vol 303, No. 7

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ORIGINAL CONTRIBUTION

Ancestry and Pathologyin King Tutankhamun’s FamilyZahi Hawass, PhD

Yehia Z. Gad, MD

Somaia Ismail, PhD

Rabab Khairat, MSc

Dina Fathalla, MSc

Naglaa Hasan, MSc

Amal Ahmed, BPharm

Hisham Elleithy, MA Markus Ball, MSc

Fawzi Gaballah, PhD

Sally Wasef, MSc

Mohamed Fateen, MD

Hany Amer, PhD

Paul Gostner, MD

Ashraf Selim, MD

Albert Zink, PhD

Carsten M. Pusch, PhD

THE 18TH DYNASTY (CIRCA 1550-1295 BC) of the New King-dom (circa 1550-1070 BC) wasone of the most powerful royal

houses of ancient Egypt. The pharaohAkhenaten, who ruled from circa 1351to 1334 BC, is considered one of themost controversial of the Egyptian pha-raohs, because his attempt to radicallytransform traditional religion affectedall facets of society and caused greatturmoil.

Akhenaten’s eventual successor, Tut-ankhamun, is probably the most fa-

mous of all pharaohs, although histen-ure was brief. He died in the ninth yearofhisreign,circa1324BC, atage19years.Little was known of Tutankhamun andhis ancestry prior to Howard Carter’sdiscovery of his intact tomb (KV62) in

For editorial comment see p 667.

Author Affiliations: Supreme Council of Antiquities,Cairo, Egypt (Dr Hawass and Mr Elleithy); NationalResearchCenter,Cairo,Egypt(Drs Gad, Ismail,and Amer andMss Hasanand Ahmed);AncientDNA Laboratory,EgyptianMuseum,Cairo,Egypt (DrsGad andIsmail andMss Fathalla, Khairat, Hasan, andAhmed); Institute ofHuman Genetics, Division of Molecular Genetics, Uni-versity of Tübingen, Tübingen, Germany (Ms Khairat,MrBall, andDr Pusch); Learning Resource Center,Kasr Al AinyFaculty ofMedicine,CairoUniversity,Cairo, Egypt

(DrsGaballah andFateenand Ms Wasef);Departmentof Radiodiagnostics, Central Hospital Bolzano, Bol-zano,Italy (Dr Gostner); Departmentof Radiology, Kasr Al AinyFacultyof Medicine,Cairo, Egypt (Dr Selim); andInstitute for Mummies and the Iceman, EURAC, Bol-zano, Italy (Dr Zink).CorrespondingAuthor:CarstenM. Pusch,PhD, InstituteofHumanGenetics,Division ofMolecular Genetics,Uni-versityof Tübingen, Wilhelmstraße27, D-72074,Tübin-gen, Germany ([email protected]).

Context The New Kingdom in ancient Egypt, comprising the 18th, 19th, and 20thdynasties, spanned the mid-16th to the early 11th centuries BC. The late 18th dy-nasty, which included the reigns of pharaohs Akhenaten and Tutankhamun, was anextraordinary time. The identification of a number of royal mummies from this era,the exact relationships between some members of the royal family, and possible ill-nesses and causes of death have been matters of debate.

Objectives To introduce a new approach to molecular and medical Egyptology, todetermine familial relationships among 11 royal mummies of the New Kingdom, andto search for pathological features attributable to possible murder, consanguinity, in-

herited disorders, and infectious diseases.Design From September 2007 to October 2009, royal mummies underwent de-tailed anthropological, radiological, and genetic studies as part of the King Tut-ankhamun Family Project. Mummies distinct from Tutankhamun’s immediate lineageserved as the genetic and morphological reference. To authenticate DNA results, ana-lytical steps were repeated and independently replicated in a secondancient DNA labo-ratory staffed by a separate group of personnel. Eleven royal mummies dating fromcirca 1410-1324 BC and suspected of being kindred of Tutankhamunand 5 royal mum-mies dating to an earlier period, circa 1550-1479 BC, were examined.

Main Outcome Measures Microsatellite-based haplotypes in the mummies, gen-erational segregation of alleles within possible pedigree variants, and correlation ofidentified diseases with individual age, archeological evidence, and the written his-torical record.

Results Genetic fingerprinting allowed the construction of a 5-generation pedigree

of Tutankhamun’s immediate lineage. The KV55 mummy and KV35YL were identi-fied as the parents of Tutankhamun. No signs of gynecomastia and craniosynostoses(eg, Antley-Bixler syndrome) or Marfan syndrome were found, but an accumulationof malformations in Tutankhamun’s family was evident. Several pathologies includingKöhler disease II were diagnosed in Tutankhamun;none alone would have caused death.Genetic testing for STEVOR, AMA1, or MSP1 genes specific for Plasmodium falcipa-rum revealed indications of malaria tropica in 4 mummies, including Tutankhamun’s.These results suggest avascular bone necrosis in conjunction with the malarial infec-tion as the most likely cause of death in Tutankhamun. Walking impairment and ma-larial disease sustained by Tutankhamun is supported by the discovery of canes andan afterlife pharmacy in his tomb.

Conclusion Using a multidisciplinary scientific approach, we showed the feasibilityof gathering data on Pharaonic kinship and diseases and speculated about individualcauses of death.

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the Valley of the Kings in 1922, buthis mummy and the priceless trea-sures buriedwith him, along with otherimportant archeological discoveriesof the 20th century, have provided sig-nificant information about the boy pha-

raoh’s life and family.Because Tutankhamundied so youngand left no heirs, numerous specula-tionson familial disease have been made.The presence of disease is further sup-ported by numerous reliefs, statuettes,and other sculptures of Akhenaten andhis family dating from the Amarna pe-riod (circa 1353-1323 BC). These arti-facts show the royalty of that era as hav-inga somewhat androgynous appearanceor a bizarre form of gynecomastia. Spe-cific diseases that have been suggestedto explain this appearance include

Marfan syndrome, Wilson-Turner X-linked mental retardation syndrome,Fröhlich syndrome (adiposogenital dys-trophy), Klinefelter syndrome, andro-gen insensitivity syndrome, aromataseexcess syndrome in conjunction withsagittal craniosynostosis syndrome, orAntley-Bixler syndrome or a variantformofthatsyndrome.1-4However, most ofthedisease diagnoses are hypotheses de-rived by observing and interpreting ar-tifacts and not by evaluating the mum-mifiedremains of royal individuals apart

from these artifacts.To shed lighton the putative diseases

and causes of death in Tutankhamun’simmediate lineage,we firstusedmolecu-lar genetic methods to determine kin-ship within that lineage. Whereas someindividual relationships were knownfrom historical records, the identity of most of the mummies under investiga-tion wasstill uncertain. Wealso searchedspecifically for pathologies, inheriteddiseases, and causes of death. For ex-ample, many scholars have hypoth-

esizedthat Tutankhamun’s death wasat-tributable to an accident, such as a fallfrom his chariot or a kick by a horse orother animal; septicemia or fat embo-lism secondary to a femur fracture; mur-der bya blowto the backof the head; orpoisoning.5-10 We had access to mum-mies that had never before been stud-ied with the methods we used.

METHODSMummies

In addition to Tutankhamun, 10 mum-mies possibly or definitely closely re-latedin some wayto Tutankhamun werechosen for this 2-year project; of these,

the identities were certain for only 3. Inaddition to these 11 mummies, 5 otherroyal individuals dating to theearly NewKingdom were selected that were dis-tinct from the putative members of theTutankhamun lineage. These 5 mum-mies were used as a morphological (ex-cluding Ahmose-Nefertari) and genetic(excluding Thutmose II) control group.All mummies are listed inTABLE 1, andfull-body computedtomography recon-structions of the mummies are avail-able in the online feature at http://www.jama.com.

Radiology

All of the mummies, except for that of Ahmose-Nefertari,werescannedusing amultidetectorcomputedtomographyunit(Somatom Emotion 6; Siemens MedicalSolutions, Malvern, Pennsylvania) in-stalled on a truck.Thetomography unitwas used to examine the mummy of Tutankhamun and those of the 2 wom-en from tomb KV35 in Luxor as well astherest of themummies at the EgyptianMuseum in Cairo (eAppendix, avail-

able at http://www.jama.com). Ce-phalic indices ofmummy heads were de-termined according to the method of Weber et al.11

Molecular Genetics

We adopted the previously publishedcriteria for ancient DNA authentica-tion, which form a consensus outlinefor executing research studies using an-cient DNA (eAppendix).12,13 Samplingof bone tissue and DNA extraction andpurification were performed accord-

ing to protocols previously pub-lished.14,15 Negative and blank extrac-tioncontrolswereprocessed along witheach sample. In addition, water andother aqueous polymerase chain reac-tion (PCR) components were moni-tored using the sensitive internal-Alu-PCR protocol16 to assess contaminationwith modern human DNA.

Sixteen Y-chromosomal short tan-dem repeats (DYS456, DYS389I,DYS390, DYS389II, DYS458, DYS19,DYS385, DYS393, DYS391, DYS439,DY S 6 3 5 , DY S 3 9 2 , Y - G AT A- H 4 ,DYS437, DYS438, DYS448) were am-

plified according to the manufactur-er’sprotocolusing the AmpF\STR YfilerPCR amplification kit (Applied Biosys-tems, Foster City, California). The Iden-tifiler kit and the AmpF\STR Minifilerkit (Applied Biosystems) were usedfor amplification of 8 polymorphic mi-crosatellites of the nuclear genome(D13S317, D7S820, D2S1338, D21S11,D16S539, D18S51, CSF1PO, FGA).

To test for Plasmodium falciparumDNA, PCR primers were designed thatspecifically amplify small subtelo-meric variable open reading frame

(STEVOR), apical membrane antigen 1( AMA1), and merozoite surface pro-tein1(MSP1) gene fragments with sizesof 100 to 250 base pairs(bp). PCR prod-ucts and cloned DNA fragments weresequenced by the Sanger method(eAppendix). Purified amplicons wererun on a genetic analyzer (ABI Prism3130, Applied Biosystems). Microsat-ellites were interpreted with DataCollection Software version 3.0 andGeneMapper ID version 3.2 (AppliedBiosystems). Lasergene version 8.0

(DNAstar, Madison, Wisconsin) andBioEdit version 7.0.9 (Ibis Biosci-ences, Carlsbad, California) were usedto establish multisequence align-ments (eAppendix).

RESULTS

Kinship Analyses

To elucidate the genealogy in Tut-ankhamun’sfamily, microsatellitemark-ers were used to achieve genetic finger-prints of all mummies. All 8 femalestested were negative for the examined

polymorphic Y-chromosomal loci, un-derlining the specificity of the ap-proach. The repeated search for hemi-zygous Y allelesin themalesyieldedfewresults, with differingsuccess in thevari-ous markers contained in the multiplexPCR kit used. Markers DYS393 and Y-GATA-H4 showed identical allele con-stellations(repeat motif locatedin themi-

ANCESTRY AND PATHOLOGY IN KING TUTANKHAMUN’S FAMILY

©2010 American Medical Association. All rights reserved. (Reprinted) JAMA, February 17, 2010—Vol 303, No. 7 639



crosatellite allele reiterated 13 and 11times, respectively) in Amenhotep III,KV55, and Tutankhamun but differentallelotypes in the nonrelatedCCG61065sample from TT320 (9 and 9, respec-tively). Syngeneic Y-chromosomal DNA

in the 3 former mummies indicates thatthey share the same paternal lineage.

These results were repeatedly ob-tained with DNA extracted from 2 to 4different biopsies per mummy; more-over,they differed from theY profiles of the male laboratory staff and were inde-pendently reproduced twice in a sec-

ond laboratory physically isolated fromthe first, data-generating laboratory.

An up to 30-fold testing of polymor-phicautosomalmicrosatellite locivia thecombined use of the Identifiler andAmpF\STRMinifilerkits(AppliedBiosys-tems)yieldedcompletedatasetsfor all8markers in 7 mummies (Thuya, Yuya,

AmenhotepIII,Tutankhamun,KV55,andboth female mummies from KV35) but

Table 1. Characteristics of the Royal 18th-Dynasty Mummies Under Investigation (N = 16)

TombExcavator,

Discovery Date Mummy Sex Age, y Previously Supposed Identity Status, Reign Storage

KV62 H. Carter, 1922 Tutankhamun M 19 Identified, likely son of Akhenaten, Amenhotep III, or Smenkhkare

Pharaoh, circa1333-1324 BC

KV62, Luxor

Putative Members of the Tutankhamun LineageKV46 J. Quibell and T. M.

Davis, 1905 Thuya F 50-60 Identified, grandmother or great

grandmother of Tutankhamun,mother of Tiye

Noblewoman,a

circa 1410-1360 BC

Egyptian Museum,Cairo

Yuya M 50-60 Identified, grandfather or greatgrandfather of Tutankhamun,father of Tiye

Nobleman,a circa1410-1360 BC


KV55 E. Ayrton, 1907 KV55 M 35-45b Smenkhkare,Akhenaten Pharaoh,circa

1351-1334 BC

Egyptian Museum,

Cairo

KV35 V. Loret, 1898 Amenhotep III M 50 Identified, father ofAkhenaten Pharaoh,circa1388-1351 BC


KV35YL F 25-35 Nefertiti, 18th-dynasty queenor princess

Queen,a circa1370-1330 BC;noblewomana

KV35, Luxor

KV35EL F 50 Nefertiti, Tiye, 1 8th-dynasty q ueen Queen,a circa1388-1351 BC;Queen, circa1370-1330 BC

KV35, Luxor

KV62 H. Carter, 1922 Fetus 1 F 5 mo Tutankhamun’s daughter Princess Kasr El Ainy, Facultyof Medicine,Cairo University

Fetus 2 F 7 mo Tutankhamun’s daughter Princess Kasr El Ainy, Facultyof Medicine,Cairo University

KV21 G. B. Belzoni, 1817(reinvestigated byD. Ryan, 1989)

KV21A F 25-40 1 8th-dynasty q ueen o r princess Royal female,noblewoman Egyptian Museum,Cairo

KV21B F 25-40 1 8th-dynasty q ueen o r princess Royal female,noblewoman


Morphological and Genetic Control Group of 18th-Dynasty Mummies TT320 Initially discovered by

the Abd er Rassulfamily, officialdiscovery 1881;tomb cleared byE . Brugsch, 1881

CCG61065 M 30 Originallythought to be ThutmoseI, the father of Hatshepsut;long considered an unidenti-fied royal of the 18th dynasty

Royal male Egyptian Museum,Cairo

Thutmose II(CCG61066)

M 30 Identified, son of ThutmoseI,half-brother and husbandof Hatshepsut

Pharaoh, circa1492-1479 BC


Ahmose-Nefertari(CCG61055)

F 70 Identified, queen and s isterof Ahmose I, possiblygrandmother of

Hatshepsut

Queen, circa1550-1525 BC


KV60OriginaltombKV20

G. B. Belzoni, 1817(excavated byH. Carter,1903-1904)

Hatshepsut(KV60A)

F 50 Identified, Hatshepsut, daughterof Thutmose I

Queen, Pharaoh,circa 1503-1482 BC


Sitra-In(KV60B)

F 50 Identified, Sitra-In, royal wet nurseof Hatshepsut

Noblewoman, circa1540-1490 BCa


Abbreviations: EL, elder lady; KV, Valley of the Kings Thebes; TT, Theban Tomb at Deir el-Bahari; YL, younger lady.a Estimated lifetime.b The mummy in KV55 was previously thought to be in his 20s when he died. However, our new computed tomography investigation revealed that he lived to be much older.





only partial data for both KV62 fetusesand the KV21A and KV21B mummies(FIGURE 1). Repeated attempts to com-plete the profiles in the 4 latter mum-mies were not successful; however, wewereabletoreplicatesomeoftheresultsforthe previous mummies more than 4times in the second, independent labo-ratory (Figure 1). Moreover, becausethese profiles differed from those of the laboratory staff and were not iden-

ticalto theonesestablishedfor thecon-trol group, the data were consideredauthentic.

Based on the partial Y-chromosomalinformation on the amount of autoso-mal half-allele sharing and family triolikelihood calculation, the most plau-sible 5-generation pedigree was con-structed.Weidentified Yuya andThuyaas great-grandparents of Tutankhamun,Amenhotep III and KV35EL as hisgrandparents, and the KV55 male andKV35YLas hissiblingparents(Figure1,FIGURE 2

, and online interactive kin-ship analysis and pedigree; for detailson kinship statistics, see eAppendix).

Gynecomastia, Feminity,

and Syndromes

Themostprominentfeatureexhibitedbythe art of the pharaoh Akhenaten, seenalso to a lesser degree in thestatues and

reliefs of Tutankhamun, is a markedlyfeminized appearance (eFigure 1A-C),reasonably suggesting some form of gy-necomastia or Marfan syndrome as anunderlying disease.1-4 However, puta-tive breasts in Tutankhamun andhisfa-ther Akhenaten (KV55) cannot be de-

termined,because KV55 is a mummifiedskeleton and Tutankhamun lacks thefrontal part of the chest wall. The penisof Tutankhamun,whichis no longer at-tached to the body, is well developed.Furthermore, the pelvic bones of Tut-ankhamun are almost entirely missing,

Figure 1. Microsatellite Data of Mummies Thought to Belong to the Tutankhamun Kindred

Microsatellite markers

D13S317 D7S820 D2S1338 D21S11 D16S539 D18S51 CSF1PO FGA Origin of transmitted allelesbased on kinship analysis

n = number of repeat motif reiterations at locus

Amenhotep IIIn

Nontransmitted allelesn

Yuyan

Thuyan

No data obtained

11 13 6 15 22 27 29 34 6 10 12 22 9 12 20 25Yuya (KV46)

11 12 10 15 22 26 26 29 6 11 19 22 9 12 20 26KV35ELa,c

10 16 6 15 16 27 25 34 8 13 16 22 6 9 23 31 Amenhotep III (KV35)

10 12 15 15 16 26 29 34 11 13 16 19 9 12 20 23KV55b,c

10 12 6 10 16 26 25 29 8 11 16 19 6 12 20 23KV35YLc

10 12 10 15 16 26 29 34 8 13 19 19 6 12 23 23Tutankhamun (KV62)c

10 16 26 35 8 10 12 23KV21A

10 17 26 11 13 12KV21B

12 16 10 13 16 29 8 19 12 23Fetus 1 (KV62)

10 6 15 26 29 35 8 13 10 19 12 23Fetus 2 (KV62)

9 12 10 13 19 26 26 35 11 8 19 7 12 26Thuya (KV46) 13 24

The length of each microsatellite allele was determined in base pairs and converted by software into the number of actual reiterations of repeat motifs at the corre-sponding locus. All established genotypes differ from those of the laboratory staff and the ancient control group. Note that allele origins in KV21A and KV21B aresuggestive and do not serve as proof of relationship with the Amenhotep III and Thuya lineages. See online interactive kinship analysis and pedigree.a Identified as Tiye. See eAppendix for additional commentary.b

Identified as Akhenaten. See eAppendix for additional commentary.c Data replication was successfully performed in the second Cairo laboratory.

Figure 2. Pedigree Showing the Genetic Relationships of the Tested 18th-Dynasty Mummies

II

III

IV

V

I

Yuya

(KV46)Thuya(KV46)

Amenhotep III

(KV35)

Tiye

(KV35EL)a

Akhenaten(KV55)a

?(KV35YL)a

Consanguinity

Proposed relationship,insufficient data

Tutankhamun

(KV62)

Fetus 1

(KV62)

Fetus 2

(KV62)

?

(KV21A)a ?

(KV21B)a

Male

Female

Stillbirth

Double line, indicating consanguinity, here represents a first-degree brother-sister relationship. Fetus 1 and fetus2 can be daughters of Tutankhamun; however, the mother is not yet genetically identified. The data obtainedfrom KV21A suggest her as the mother of the fetuses. However, the few data are not statistically significant todefine her as Ankhensenamun. See online interactive kinship analysis and pedigree.aSee eAppendix for additional commentary on identity.





and the pelvis of KV55, which is pres-ent butfragmented, does notshow femi-nine traits after reconstruction usingcomputed tomography (eAppendix,eFigure 1D-G, and online interactivefeature).

Oneof theobviousfeatures ofMarfansyndrome is dolichocephaly.17-19 Withthe exception of Yuya (cephalic index,70.3), none of the mummies of the Tut-ankhamun lineage has a cephalic indexof 75 or less (ie, indicating dolicho-cephaly). Instead, Akhenaten has an in-dex of 81.0 andTutankhamun an indexof83.9, indicating brachycephaly. Fromthe control group, Thutmose II and theTT320-CCG61065 mummy show doli-chocephaly, withcephalicindicesof73.4and74.3,respectively.Becausethereisnosignofprematureclosureofsutures,none

of the skull shapes can be consideredpathological. The complex diagnosis of Marfansyndromeisbasedoncertaincom-binationsofmajorandminorclinicalfea-tures.18 Following this classification, aMarfandiagnosiscannot besupportedin

thesemummies.(TABLE2

).Antley-Bixlersyndrome is also excluded in Tut-ankhamun andAkhenaten because theirbrachycephaly is not attributable to cra-niosynostoses,andfurthersignsofAntley-Bixlerorothersyndromesaremissingorunspecific.

Pathology in the Royal Mummies

Tutankhamun’s mummy was exam-ined several times radiologically.20-23

Our inspection of the skull and trunkdid not reveal novel information, butdetailed examination of the king’s feet

yielded new data. Compared with thenormal anatomy of the foot (FIGURE3),the right foot had a low arch (Rocherangle, 132°; normal value, 126°). Themedial longitudinal arch of the left footwasslightly higher thannormal (Rocher

angle, 120°) (FIGURE 4

A), with thefore-foot in supine andinwardly rotated po-sition akin to an equinovarus foot de-formity (Figure 4B). There were nopathological findingson the bonestruc-ture of the right metatarsal heads(FIGURE 5A). In contrast, the left sec-ond metatarsal head was strongly de-formed and displayed a distinctly al-tered structure, with areas of increasedand decreased bone density indicatingbone necrosis (Figure 5B). The studyfurther showed a widening of the sec-ond metatarsophalangeal joint space,

Table 2. Evaluation of Marfanoid Features in the Collection of Royal 18th-Dynasty Mummies Under Investigationa

MarfanoidFeatures

Tutankhamun Lineage Control Group

Tut-ankh-amun(KV62)

Thuya(KV46)

Yuya(KV46)

Akhenaten(KV55)

Amen-hotep III(KV35) KV35YL

Tiye(KV35EL)

Fetus 1(KV62)b

Fetus 2(KV62)b KV21A KV21B

TT320-CCG61065

ThutmoseII

(TT320-CCG61066)

Hat-shep-

sut(KV60A)

Sitra-In(KV60B)

Major criteriaDolichostenomelia − − − − − − − − − − − − − − −

Body height,cm

167 150 166 160 160 158 145 27.5 38 150 150 157 173 153 151

Scoliosis,kyphoscoliosis

c

d −

c − c

c −

c

c

c

c

c − c

Pectus carinatum NA − − NA NA − − NA NA NA NA − NA − −

Pectus

excavatum

NA − − NA NA − − NA NA NA NA − NA − −

Acetabularprotrusion

NA − − − − − − NA NA − − − − − −

Pes planus c,e − − NA − NA − NA NA − − − − − −

Minor criteriaDolichocephaly − −

c,f − − − − NA NA NA NA c,f

c,f − −

Arachnodactyly − −g −g NA NA − −g − − − − NA − − −

Mandibularretrognathism

− − NA NA NA NA − −

Hypoplasia of cheek bones

− − − − − NA NA NA NA − − − −

Highly archedpalate

− − − − NA NA NA NA − − − −

Crowding of teeth − − − − NA NA NA NA − − −

Striae atrophicae − − − NA NA − − NA NA NA NA − − − −

Mitral annularcalcification

NA − − NA NA NA NA NA NA NA NA − − NA

CCA feature(rumpled helixof the ear)

− − − NA NA − NA NA NA NA − −

Abbreviations: CCA, congenital contractural arachnodactyly; EL, elder lady; minus symbol, feature absent; NA, not available (certain parts of the mummiesare missingor fragmented, ie,feature cannot be observed); plus symbol, feature present; YL, younger lady.

a No computed tomography scan was performed on the mummy of Ahmose-Nefertari (TT320-CCG61055).b Many of the features cannot be evaluated in fetuses.c Mild form.d Cobb angle in Thuya, 20° (severe form of scoliosis or kyphoscoliosis); in all other individuals, 20° (mild form).e Pes planus in Tutankhamun is not caused by a medial displacement of the inner malleolus (ie, no marfanoid flatfoot).f Shape of the head is of natural occurrence, ie, not caused by craniosynostosis syndromes.g Madonna fingers.





with a normal articulatingsurface of theproximal phalanx. The third metatar-sal head was only slightydeformed; thebony structure, however, showed signsof bone necrosis. The remaining leftmetatarsal heads appeared to be of nor-

mal structure (Figure 5B). The plan-tar surface of the left second metatar-sal head shows a crater-shaped boneand a soft tissue defect in the area of bone necrosis (Figure 5C). The sec-ond and third toes on the left foot arein abduction. The second toe isshortened because it lacks the middlephalanx (oligodactyly [hypophalan-gism]). The proximal phalanx di-rectly articulates with the distal pha-lanx (Figure 5D).

Except for Ahmose-Nefertari, all re-maining mummies were subjectedto ra-

diological analyses. Along with vari-ous bony malformations (eg, cleftpalate, kyphoscoliosis, clubfeet, flatfeet) in the remaining mummies, indi-cations of bone degeneration, neoplas-tic changes, and trauma were alsofound. These various findings are listedinTABLE3 andare described in theeAp-pendix.

Infectious Diseases

Various infectious diseases are sus-pected or known to have been preva-

lent in antiquity,24-27

and some are de-scribed in remarkable detail in Egyptianpapyri (eg, Papyrus Ebers, circa 1520BC). Positive results were not found forpandemic plague (Black Death, bu-bonic plague), tuberculosis, leprosy, orleishmaniasis, but we identified DNAof P falciparum (the malaria parasite)in several of the royal mummies. Am-plification of the P falciparum STEVORgene family28 repeatedly yielded 149-bpand 1 8 9 - b p am pl i co ns fo r T ut-ankhamun and the TT320-CCG61065

mummy and also yielded a faint PCR band using DNA of the Yuya mummy.This result was replicated in furtherPCRs using DNA from other biopsies(for details on STEVOR data see eAp-pendix and eFigure 2).

To consolidate or disprove this re-sult, we targeted a further Plasmodiumgene using new DNA extracts from the

royal mummies in our study. We iden-tified4 mummies as positive for AMA1,a merozoite protein responsible for thesuccessful binding of the parasite to theerythrocyte membrane, by amplifyingDNA fragments locating to the con-

served region of the AMA1 gene(FIGURE 6). The AMA1 PCR frag-ments were obtained for all mummiestesting positive in the earlier STEVORassays(ie,Tutankhamun, Yuya, TT320-CCG61065). In addition, we also ob-tained a positive typing for Thuya. Rep-etition of these experiments in thesecond laboratory using DNA extrac-

tions from new biopsies confirmed theprevious data (Figure 6; for details on AMA1 data, see eAppendix).

In addition to theSTEVOR and AMA1genes, we attempted amplification of alleles of theMSP1 and MSP2 genes spe-

cificto P falciparum. Because of thefrag-mented nature of the ancient DNA, wedid not obtain positive amplificationswhen targeting the larger (400 bp)PCR alleles of the MSP2 gene but weresuccessful in amplifying differentallelesof the MSP1 gene (for details on MSP1data, see eAppendix).29,30 Using ex-tractsfrom Tutankhamun andYuya,we

Figure 3. Normal Foot Anatomy

Medial cuneiform

Cuboid

Intermediate cuneiform

Lateralcuneiform

Metatarsals

Proximal phalanges

Middle phalanges

Distal phalanges

Calcaneus Calcaneus

Talus

Talus

NavicularMedial cuneiform

Navicular

Cuboid

M EDIAL V IEW

SUPERIO R V IEW

Figure 4. Analysis of Malformations in the Feet of Tutankhamun

A Sagittal CT reconstruction Axial CT cross sectionsB

120°

132°

L

R

R L

A, As indicated by the angle between the axis of the first metatarsal and the line between the lowest point ofthe calcaneal tuberosity to the lowest point of the calcaneocuboid articulation (Rocher angle), the arch of theright footis flat(132°)comparedwiththat ofthe left (120°). The Rocherangle ofa normalfootis 126°. B,Thesupine and inwardly rotated position of the left foot are further features of clubfoot.





repeatedly amplified the RO33 andMAD20 alleles, which is indicative of at least a doubleinfection with the P fal-ciparum parasite. The DNA of Thuyayielded amplicons for the RO33 allele.The DNA of TT320-CCG61065 wasre-

fractory to MSP1 amplifications. Clon-ing the obtained allelic fragments intoTA plasmid vectors and subsequentSanger sequencing of 21 clones desig-nated the sequences as specific forMSP1(eAppendix).

COMMENTKinship Determination

More than 55 bone biopsies were usedto elucidate the individual relation-ships of 18th-dynasty individuals, withthe result that several of the anony-

mous mummies or those with sus-pected identities are now able to beaddressed by name. These includeKV35EL, who is Tiye, mother of Akhenaten and grandmother of Tut-ankhamun,andtheKV55 mummy, who

is most probably Akhenaten, father of Tutankhamun (Figure 2, eAppendix,and online interactive kinship analysisandpedigree). Thelatter kinship is sup-ported in that several unique anthro-pological features are shared by the 2

mummies and that the blood group of both individuals is identical.31,32

Disease or Amarna Artistic Style?

Macroscopic and radiological inspec-tion of the mummies did not showspecific signs of gynecomastia, cra-niosynostoses, Antley-Bixler syn-drome or deficiency in cytochromeP450 oxidoreductase, Marfan syn-drome, or related disorders (eAppen-dix, Table 2). Therefore, the particu-lar artisticpresentationof persons in theAmarna period is confirmed as a roy-

ally decreed style most probably relatedto the religious reforms of Akhenaten.It is unlikely that either Tutankhamunor Akhenaten actually displayed a sig-nificantly bizarre or feminine physique.

It is important to note that ancientEgyptian kings typically had them-selves and their families represented inan idealized fashion. A recent radio-graphic examination of the Nefertitibust in the Berlin Museum illustratesthis clearly by showing that the origi-nal face of Nefertiti, present as a thin

layer beneath the outer surface, is lessbeautiful than that represented by theartifact.33 Differences include the anglesof the eyelids, creases around the cor-ners of the mouth on the limestone sur-face, and a slight bump on the ridge of the nose.34 Thus, especially in the ab-sence of morphological justification,Akhenaten’s choice of a “grotesque”style becomes even more significant.

Walking Impairment and Canes

Tutankhamun had a juvenile aseptic

bone necrosis of the left second andthird metatarsals (Köhler disease II,Freiberg-Köhler syndrome). The wid-ening of the metatarsal-phalangeal jo in t sp ac e, as we ll as se co nd ar ychanges of the second and third meta-tarsal heads, indicate that the diseasewas still flourishing at the time of death.35 Bone and soft tissue loss at

Figure 5. Analysis of Pathology in the Feet of Tutankhamun

Axial cross sections of right foot A

R

Sagittal CT reconstructionthrough second metatarsals

C Reconstruction of left and right feetD

L

RL

Axial cross sections of left footB

A, The heads of all metatarsal bones as well as metatarsal phalangeal articulations of the right foot are clearlydiscernable and completely preserved. B, In the left foot, the second metatarsal bone head (yellow arrow-heads)shows signs of bone necrosis accompanied by anterior displacementof thesecond toeand widening ofthe second metatarsophalangeal joint space (white arrowheads). The third metatarsal bone head is similarlydeformed (blue arrowheads), displaying features of bone necrosis as well. Metatarsal bone heads 1, 4, and 5arenormal in sizeand structure. C, Theright foot shows no pathologicalfindings.The secondmetatarsal bonehead shows evidence of necrosis with loss of bone substance and soft tissue (yellow arrowhead). The secondtoe of the left foot lacks the middle phalanx (oligodactyly [hypophalangism], black arrowhead). D, The rightfoot shows no pathological findings. In the left foot, the second metatarsal head is necrotic (yellow arrow-head) and the second toe is missing the middle phalanx (oligodactyly [hypophalangism], black arrowhead), isanteriorly displaced, and the distal phalanx is subluxated.





the second metatarsal phalangealarticulation could further indicatethat an acute inflammatory conditionwas present on the basis of an ulcer-ative 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 theright foot, causing flattening of thefoot arch (pes planus).

There is evidence that Tutankhamunmay have had this impairment for quitesome time. The walking disability canbe substantially aided by the use of acane. Howard Carter discovered 130whole andpartialexamplesof sticksandstaves (eFigure 3A) in the king’s tomb,supporting the hypothesis of a walk-

ing impairment.

36

Traces of wear canbe seen on a number of thesticks, dem-onstrating that they were used in theking’s lifetime (eFigure 3B). Addi-tional evidence for some sort of physi-cal disability is found in a number of 2-dimensional images from Tut-ankhamun’s reign that show himseatedwhile engaged in activities for which he

normally should have been standing,such as hunting (eAppendix andeFigure 3C).37,38

Malaria Tropica

Macroscopic studies revealed areas of patchy skin changes on the pharaoh’s

Table 3. Anomalies and Diseases in This Collection of 18th-Dynasty Mummies

Mummy Malformations Other Pathologies and Diseases

Tutankhamun (KV62) Cleft palate, mild clubfoot left, pes planusright, oligodactyly (hypophalangism)right, mild kyphoscoliosis

Leg fracture of type 33C3, a Köhler disease II, malariatropica

Tutankhamun kindred Thuya (KV46) Severe kyphoscoliosis Dental granuloma, arteriosclerosis, incisional hernia,

malaria tropica Yuya (KV46) None Dental granuloma, incisional hernia, malaria t ropica

Akhenaten (KV55) Cleft palate, scoliosis Osteoma in the maxillary sinus, femoral osseouscollapse, or bone fibroma

Amenhotep III (KV35) Clubfoot Caries, dental granuloma, bony erosions 2-3 cm at theinner table and diploe of the right parietal bone,spondylarthrosis

KV35YL Scoliosis Traumatic events (face, calvarium)b

Tiye (KV35EL) Mild scoliosis Struma, incisional hernia

Fetus 1 (KV62) None Stillbirth

Fetus 2 (KV62) Mild scoliosis Stillbirth

KV21A Scoliosis, clubfeet None

KV21B Scoliosis, clubfoot None

Control Group TT320-CCG61065 Kyphoscoliosis, pelvic obliquity Incisional hernia, malaria tropica, arrow wound to chest b

Thutmose II (TT320-CCG61066) Kyphoscoliosis, hollow feet ( pes cavus) Calcified heart valves, incisional hernia

Hatshepsut (KV60A) None Dental abscessa, incisional hernia, slipped disk (L5-S1),putative metastatic cancer left iliac bone b

Sitra-In (KV60B) Kyphoscoliosis Incisional hernia

Abbreviations: CCG, Cairo Catalogue General; KV, Valley of the Kings Thebes; TT, Theban Tomb at Deir el-Bahari.a AO classification.b Assumed cause of death.

Figure 6. Identification of Plasmodial DNA in 18th-Dynasty Mummies

A m e n

h o t e

p I I I ( K V

3 5 )

K V 5 5

b

F e t u

s 1 ( K V

6 2 )

F e t u

s 2 ( K V

6 2 )

K V 2 1

A

K V 2 1

B

D N A

m a r

k e r

K V 3 5

E L c

K V 3 5

Y L

H a t s

h e p s

u t ( K V

6 0 A )

A h m o s

e - N e

f e r t a

r i

( T T 3 2

0 - C C

G 6 1 0

5 5 )

A h m o s

e - N e

f e r t a

r i

( T T 3 2

0 - C C

G 6 1 0

5 5 )

A m e n

h o t e

p I I I ( K V

3 5 )

K V 5 5

b

K V 2 1

A

K V 2 1

B

D N A

m a r k e

r

K V 3 5

E L c

K V 3 5

Y L

F e t u

s 1 ( K V

6 2 )

F e t u

s 2 ( K V

6 2 )

H a t s

h e p s

u t ( K V

6 0 A )

S i t r a

- I n ( K V

6 0 B )

196 bp

196 bp

T u t a

n k h a

m u n

( K V 6 2

) a

+

T u t a

n k h a

m u n

( K V 6 2

) a

+

T h u y

a ( K V 4 6

)

+

Y u y a

( K V 4 6

)

+

S i t r a

- I n ( K V

6 0 B )

T T 3 2

0 - C C

G 6 1 0

6 5

+

T u t a

n k h a

m u n

( K V 6 2

) a

+

T u t a

n k h a

m u n

( K V 6 2

) a

+

T h u y

a ( K V 4 6

)

+

Y u y a

( K V 4 6

)

+

T T 3 2

0 - C C

G 6 1 0

6 5

+

C o n t

r o l

( n e g a t

i v e )

C o n t

r o l

( n e g a t

i v e )

A

B

A, Polymerase chain reaction amplification of a 196–base pair (bp) apical membrane antigen 1 ( AMA1) frag-ment of Plasmodium falciparum in Egyptian mummies. DNA marker indicates molecular size marker phiX/

174 HaeIII. Successful amplification is indicated by “

.” B, Independent replication of the AMA1 data shownin panel A.aDifferent DNA extractions.b Identified as Akhenaten. See eAppendix for additional commentary.c Identified as Tiye. See eAppendix for additional commentary.





left cheek and neck of uncertain anam-nesis, possibly indicating an Aleppoboil, a plague spot, an inflamed mos-quito bite, or a mummification arti-fact.39 However, the genetic identifica-tion and typing of plasmodial DNA in

Tutankhamun, Thuya, Yuya, andTT320-CCG61065 showed that theymust have hadmalariatropica,themostsevere form of malaria (eAppendix).

Literary evidence for malaria infec-tion dates back to the early Greek pe-riod, when Hippocrates described theperiodic fever typical of this disease.40

Although it is believed that malariawidely affected early populations be-fore Hippocrates,27,41 until now only 1reportusing immunological tools42 andfew molecular genetic studies haveclearly identified P falciparum in an-

cient specimens.43-46 We not only iden-tified this parasite in our sample but alsoobservedindividual differences in someof the gene sequences as well as differ-ent MSP1 allele constellations in the 4positive mummies. The diversity of plasmodial DNA (ie, variability in thegenes’ base order, length polymor-phisms, or both) is a well-known phe-nomenon; however, some of the basedeviations were not found in currentDNA databases. Further research is re-quired to typify these alterations in

more detail and to assign these poten-tially unknown patterns to ancientEgyptian Plasmodium strains that dateback to 3300 to 3400 years beforepresent.

To our knowledge, this is the oldestgenetic proof for malaria in preciselydated mummies. When the infectionoccurred, its severity, and whether itcould have caused the death in the 4mummies testing positive is not known.Preliminary data show that Tut-ankhamun and Yuya had multiple in-

fections, as could be seen by the pres-ence of the 2 P falciparum allelesMAD20 and RO33 of the MSP1 in theextracts. In contrast, andtaking only theMSP1 test system into account, Thuyawas infected by only 1 strain, which dis-played the RO33 allele.

To date, no association has beenfound between P falciparum MSP1

genotypesand the clinical status of per-sons affected.47 We note that mixed P falciparum infections were detected inup to 78% of a contemporary sam-pling, and even isolates from sympto-matic children contained more than 1

Plasmodium clone.

47,48

Thus, multipleinfections appear to be the norm ratherthantheexception. Moreover, theMSP1allele frequencies tend to vary largelyin different, sometimes even neighbor-ing, areas butalso over time.29 Thus, theprevalence rate of infection is notknown—nor is it known if malaria wasan epidemic or an endemic disease andhow widely it was distributed in an-cient Egypt.

Unfortunately, there is also no dis-tinct evidence in ancient Egyptian textsof treatments for malaria, and there are

no references to the fevers and chills as-sociated with the disease.49 However,the NileDelta and the fringes ofthe NileValley were marshy areas and thus ex-cellent breeding grounds for the mos-quito genus Anopheles. Interestingly,mosquitoes are mentioned in at least 1ancient text,50 and it has also been sug-gested that the wooden frame of QueenHetepheres (fourth dynasty) served asthe support for a mosquito net.50

Herodotus also mentions that LowerEgypt was infested with mosquitoes or

other insects and that people slept un-der nets to avoid them.51 Since there isnothing in the historical or archeologi-cal record that speaks against thewide-spreadpresence of this carrier in Phara-onic times,there is no evidence that canbe used to argue against the diagnosisof malaria.

Cause of Death

Caution must be taken when interpret-ing cause of death in these mummies.It can be speculated that Yuya and

Thuya had malaria, but it is not knownif this was lethal (Table 3). Surpris-ingly, both individuals had reached anadvanced (for thetime) age of approxi-mately 50 yearsor older (Table 1). Thismeans either that the infection tookplace quite late in their lifetime, thatthey enjoyed strong genetic fitness, orthat they aquired a partial immunity

against the pathogen during their lives.Not every person infected with P falciparum becomes gravely ill, and this isespecially truein populations that havebeen exposed to malariapathogens overlong 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 immunizationmay have contributed to their survival.

On the other hand, Tutankhamunhad multiple disorders, and some of them might have reached the cumula-tive character of an inflammatory, im-mune-suppressive—and thus weaken-ing—syndrome (Table 3). He might beenvisioned as a youngbut frail king whoneeded canes to walk because of thebone-necrotic and sometimes painfulKöhler disease II, plusoligodactyly(hy-

pophalangism) in the right foot andclubfoot on the left. A sudden leg frac-ture23 possibly introduced by a fallmight have resulted in a life-threaten-ing condition when a malaria infec-tion occurred. Seeds, fruits, and leavesfound in the tomb, and possibly usedas medical treatment, support this di-agnosis (eAppendix, eFigures 3D and3E).24,25,53-57

In conclusion, this study suggests anew approach to research into the mo-lecular genealogy and pathogen paleo-

genomics of thePharaonicera. With ad-ditionaldata,a scientific discipline calledmolecular Egyptology might be estab-lished and consolidated, thereby merg-ing natural sciences, life sciences, cul-tural sciences, humanities,medicine, andother fields.

Author Contributions: Drs Hawass, Gad, Zink, andPusch had full access to all of the data in the studyandtake responsibility forthe integrity of thedata andthe accuracy of the data analysis. Study concept and design: Hawass, Gad, Zink,Pusch.Acquisition of data: Hawass, Gad, Ismail, Khairat,Fathalla, Hasan, Ahmed, Elleithy, Gaballah, Wasef,Fateen, Amer, Gostner, Selim, Zink.Analysis and interpretation of data: Hawass, Gad,

Ismail, Khairat, Fathalla, Hasan, Ball, Wasef, Fateen,Amer, Gostner, Selim, Zink, Pusch.Draftingof themanuscript: Hawass, Gad, Zink, Pusch.Critical revision of the manuscript for important in-tellectual content: Hawass, Gad, Ismail, Khairat,Fathalla, Hasan, Ahmed, Elleithy, Ball,Gaballah, Wasef,Fateen, Amer, Gostner, Selim, Zink, Pusch. Statistical analysis: Ball, Gostner, Zink, Pusch.Administrative, technical, or material support: Hawass,Gad, Ismail, Hasan, Ahmed, Elleithy, Ball, Gaballah,Fateen, Amer, Selim, Zink, Pusch. Study supervision: Gad, Ismail, Zink, Pusch.





Financial Disclosures: None reported.Funding/Support:This studywas supportedby theDis-covery Channel and the Brando Quilici productiongroup. Funding was also obtained from the Mini-Graduiertenkolleg Tübingen and the DAAD (GERLSexchange program). Siemens Medical donated ma-terial and installed the multislice computed tomogra-phy scanner used in the study.Role of the Sponsor: The funding organizations had

no role in the design and conduct of the study; thecollection, analysis, and interpretation of the data; or the preparation, review, or approval of the manu-script.Online-Only Material: The eAppendix, eFigures 1through 3, and the online feature showing full-bodycomputed tomography reconstructions of the mum-mies and interactivekinship analysis and pedigree areavailable at http://www.jama.com.Additional Contributions: Jessica Sherry, BS (man-ager, firstlaboratory),and Milena Gozzo, BDM (man-ager, second laboratory),contributedexpertmanage-ment of theancientDNA laboratoriesin Cairo,Egypt.Neither of these individuals receivedcompensation for their contributions beyond their salaries.

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Ancestry and Pathology in King Tutankhamun’s Family. JAMA, February 17, 2010—Vol 303, No. 7