King Tutankhamun: Y-DNA Haplogroup R1b1a2

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.


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

Kinship Analyses
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.


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

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).

Gynecomastia, Feminity, and Syndromes
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.