Reasons for retesting the Tutankhamun family DNA

DNA analysis involves the study of a person’s genes – i.e. their genetic inheritance. Each gene occurs in different forms called alleles. For each genetic trait there are two alleles, with one allele inherited from each parent. So parents and their offspring share alleles, as do siblings. In other words, two related people will share more of the same alleles than two unrelated people. The greatest sharing of alleles occur in cases of incest, where family members intermarry, and so their offspring will share a greater number of alleles then normally expected. This is now known to lead to health problems and infertility, but in ancient times the genetic risks were not understood, and incest was frequently practiced in order to restrict royal power to within a small group of elites.

Taking DNA samples from ancient mummies can potentially help to confirm levels of relatedness.

In 2010, the Journal of the American Medical Association (JAMA) published the findings of The Tutankhamun Family Project (Hawass et al.) after a detailed examination of eleven royal mummies: Tutankhamun; five mummies thought to be related to him; and a control group of five royal mummies from an earlier period for comparison (for a report on this work, see AE59). Using genetic fingerprinting, the team revealed a five-generation pedigree of Tutankhamun’s immediate lineage, with his parents identified as the KV55 male (declared to be Akhenaten) and the ‘Younger Lady’ in KV35.

There has been considerable debate over the methods and conclusions of this study. As a retired dentist with a particular interest in forensics, DNA, and human anatomy, Joseph Thimes reanalysed the details of this paper. He believes his findings merit a revision of this family’s interconnectivities, and calls for further testing with a wider cohort of royal mummies.

The JAMA genealogy shows a direct line of ancestry from Yuya and Thuya (parents of Queen Tiye who are not genetically related to each other) down to the two stillborn girls found in KV62. Looking at the DNA charts on which this is based, and if we take the traditional ‘top down’ look going from parent to child, this table looks perfectly normal. However, if we review this DNA tabulation laterally, as in certain in-laws and brother-to-sister associations, unexpected and possibly close affinities can be detected.

Levels of Consanguinity

One very unusual DNA pattern of concern is the full brother-sister relationship of the KV55 male to the Younger Lady (i.e. the parents of Tutankhamun). They share 11 out of 16 alleles, which indicates a high level of consanguinity within previous generations – past members of the family were possibly close cousins or even more incestuously related. The parents of KV55 and the Younger Lady were Amenhotep III and Queen Tiye, who were not full brother and sister. However, a second DNA pattern showing a 6 out of 16 allele match was found for the father-in-law/son-in-law association of Yuya and Amenhotep III. This indicates both of these individuals were probably closely related.

It is possible that Mutemwiya, a minor wife of Thutmose IV and the mother of Amenhotep III, could be the full sister, or even half-sister (sharing a mother or father), of Yuya. If a full sister, this could explain the 11/16 allele match between KV55 and the Younger Lady, but also the match between Yuya and Amenhotep III. Another possibility could be that Amenhotep II was father to both Thutmose IV and Yuya. If so, this would mean Yuya and Thutmose IV were full, three-quarter or half-brothers. This too would explain much in the high allele match of KV55 to the Younger Lady and also that of Yuya to Amenhotep III. If neither of these relationships turns out to be true, then I would suspect either a fatherdaughter relationship between Amenhotep III and Sitamun, or mother-son incest between Tiye and Crown Prince Thutmose.

It is also worth considering that Tiye shared 3 of 16 alleles with her consort, Amenhotep III – these alleles were inherited from her father, Yuya, suggesting that Amenhotep III had also inherited his alleles from Yuya – so Amenhotep III and Tiye could possibly be cousins. Also, Tiye shared 6/16 alleles with her grandson Tutankhamun, who shared 7/16 alleles with his grandfather Amenhotep III. These levels are greatly elevated from the normal 25% shared between grandparents and grandchildren, emphasizing a greater degree of closeness within the Tutankhamun family and between the royal family and the Akhmim family of Yuya.

Re-tests and More Mummies

To try to resolve some of this uncertainty, I would endorse a reexamining of all eleven original JAMA mummies, plus three other accessible mummies: the KV35 boy (found between the Younger and Elder Ladies), Amenhotep II and Thutmose IV. This re-test should be carried out using a DNA kit that produces complete – not partial – DNA profiles. I would also propose evaluating these mummies for mitochondrial DNA [which is inherited from the mother and so used to determine the maternal line of descent]; for the autosomal DNA [from chromosomes other than the X and Y sex chromosomes]; and for the amelogenin DNA [involved in the formation of tooth enamel] which is only used to determine sex. Since this retesting is more complex than pure DNA research, we would need additional expertise in areas including age resolution forensics, medical image interpretation and other disciplines possibly including dental science.

Thutmose IV

Why include the mummy of Thutmose IV in further testing? Firstly the mummy itself is accessible; but more importantly, DNA analysis of this king may help to shed light on the genetic ancestry of his wife Mutemwiya, who Aldred suggested could be Yuya’s full sister. We do not have her mummy, but if we obtain good and full DNA results from the Thutmose IV and Amenhotep III mummies, we can derive one-half of Mutemwiya’s autosomal DNA, since any of Amenhotep III’s alleles not shared with Thutmose IV must have come from his mother. We should then compare the autosomal DNA Amenhotep III inherited from his mother to that of Yuya. If we get a 50% match (approximately) then we have the possibility that Mutemwiya was Yuya’s full sister (as in the diagram opposite, top). Also, Amenhotep would have inherited his mitochondrial DNA from his mother. If this mitochondrial DNA is a match to that of Yuya, it will show that Mutemwiya has the same maternal line of descent as Yuya. This would explain both the 6/16 allele equivalency between Yuya and Amenhotep III and also the 11/16 allele match of KV55 to the Younger Lady. Of course, the DNA retesting could show no relationship at all between Mutemwiya and Yuya. Instead, if Mutemwiya’s autosomal DNA suggests that she is the full sister to Thuya (Yuya’s wife) and if the mitochondrial DNA shows the maternal line is the same between Mutemwiya and Thuya, this would be able to explain the 11/16 allele equivalency between KV55 and the Younger Lady. However, this would not explain the 6/16 alleles matching between Yuya and his son-in-law Amenhotep III. Possibly, Mutemwiya may have engineered the marriage of Amenhotep III to Tiye (especially if she were sister either to Yuya or to Thuya).

Amenhotep II

As the mummy of Amenhotep II is also available, I would suggest it to be included in any DNA retesting of the Tutankhamun family. We know that Amenhotep II was the father of Thutmose IV, but could he also have been father to Yuya? We should compare the results of the autosomal DNA on all three of these mummies, which should show if Amenhotep was indeed the father of both. A 50% match between the two sons would indicate Thutmose IV and Yuya were full brothers; a 25% match would suggest they were half-brothers. Also, if the mitochondrial DNA is equal for Yuya and

Thutmose IV, then they would probably be three-quarter brothers [sharing the same father, with different, but fullsister mothers]. In this case, a 37.5% DNA match should occur.

At this time, I do not believe that Yuya and Thutmose would be full brothers, as this may have led to a problem with succession to the throne. If they were three-quarter brothers, this means that Amenhotep II married two full sisters – one being the mother of Yuya and the other being mother to Thutmose IV. If the mitochondrial DNA did not match but the autosomal DNA demonstrates that Amenhotep II is the actual father to both of these men, then Yuya and Thutmose IV would be half-brothers. Of course, the DNA research could show there was no relationship between Thutmose IV and Yuya, in which case Amenhotep II could not have been Yuya’s father.

If Amenhotep II is the father to both Thutmose IV and Yuya, this would help explain both the high percentage of 6/16 shared alleles between Yuya and Amenhotep III and also the high percentage of 11/16 shared alleles between KV55 and the Younger Lady. These percentages can also be explained by a combination of Amenhotep II as father to Yuya and Thutmose IV, with Mutemwiya being the full or half-sister of either Yuya or Thuya.

Alternative Relationships

If the retesting shows no relationship between Mutemwiya to either Yuya or Thuya, and also no relationship between Yuya and Thutmose IV, then we would have to consider a mother-toson or father-to-daughter incest to explain the 11/16 shared alleles between KV55 and the Younger Lady. We know that Queen Tiye was neither full sister nor half-sister to Amenhotep III. We also know that in Year 30, Amenhotep III made his eldest daughter Sitamun his great royal wife. If father and daughter were the parents of the Younger Lady, who was born around Year 31, then this Younger Lady could have conceived Tutankhamun around Year 8 or 9 of Akhenaten’s reign. Such a level of incest is possible. Amenhotep married two of his daughters (Sitamun and Iset), as did Akhenaten. Ramesses II is known to have married at least three of his daughters – one (Bintanath) bore a child. The other (more unlikely?) possibility is that Queen Tiye and Crown Prince Thutmose were the parents of the Younger Lady, who must have been born before the end of Year 30 of Amenhotep III’s reign (we know that Thutmose survived to Year 29 or 30). Either one of these options could be possible, but we probably would not be able to prove which one.

Evidence from Age of Death

Assessing the approximate age of each of the royal mummies can also help with identification. Of the fourteen mummies, I would propose using lifespan differentiation methods on the KV55 male, the Younger Lady, the KV35 boy and the KV21A and B mummies [thought to be two Eighteenth Dynasty queens]. The 2010 study suggested that KV21A was the biological mother of the two foetuses, and so could be Akhesenamun. This forensic age resolution methodology should include analysis of teeth present (which works to about 21 years) and long bone fusion (which works up to the age of about 25). If these two approaches do not result in a close correlation of age for each mummy, the appropriate cranial sutures or the pubic symphysis method [which analyses wear on the joint at the front of the pelvis] should then be looked at.

The Younger Lady

I have determined that the Younger Lady lived for only 15-16 years since her third molar root formation was barely beginning to form and because her proximal radius bone head was fused. I place her birth to about Year 30 or 31 of Amenhotep III’s reign and her demise to about Year 9 of Akhenaten’s rule – possibly at the time she gave birth to Tutankhamun. To simplify this case, please note that I am assuming that Tutankhamun took over kingship of Egypt upon Akhenaten’s death. The Younger Lady is far too young to be Nefertiti and is not Meritaten nor even Baketaten since both of these princesses would only be about eight or nine years old when Tutankhamun was born. The Younger Lady could be Nebetah or an unidentified full daughter of Amenhotep III and Queen Tiye.

The KV55 Body

As the estimated range of age for the male in KV55 is 18 to 45 years, this mummy would be the ideal case to inspect using longevity determining forensics. From KV55’s mummy, we have an excellent skull with plenty of teeth and excellent bones to evaluate long bone mergers. If these two methods suggest an age of about eighteen to twenty at death, we can stop here. As Akhenaten reigned for seventeen years and produced his first daughter in his first year as pharaoh, he would have been far older than twenty at time of death; the KV55 body in this case could not be Akhenaten. If KV55’s longevity approximates to twenty-five years, then I would suggest using the cranial sutures procedure on his skull or the pubic symphysis method to get an approximate age. This might give a lifespan for the KV55 mummy life of about 25, 35, or 45 years of age. Until this further work has been carried out, we cannot know the identity of the KV55 body – possibilities include Smenkhkara, Akhenaten, Crown Prince Thutmose, or an unidentified full son of Amenhotep III and Queen Tiye.

Mother of Tutankhamun

As the KV21A mummy is headless, we would have only the long bone fusion method accessible to us. As such, we could only date this mummy up to the age of about twenty-five. The percentage of alleles shared by the KV21A body and Foetus 2 strongly suggests that KV21A is the mother of Foetus 2. The JAMA DNA information suggests it is very likely that Tutankhamun was the father of both of the still-born girls. We know that Tutankhamun married Ankhesenamun, who was the third daughter of Akhenaten and Nefertiti; so it is possible that she is mummy KV21A. Tutankhamun is not known to have had any other wives or concubines, but we cannot rule out this possibility.

If the ancient Egyptian records are accurate as to how some of the Tutankhamun family interconnect, then my principle “If this, then not that”, is significant. From the DNA patterns in JAMA, since neither KV55 nor the Younger Lady share KV21A’s alleles 35 and 10, neither of them can be her parents. If KV55 is Akhenaten, then KV21A is not Ankhesenamun. If KV21A is Ankhesenamun, then KV55 is not Akhenaten. Are the ancient Egyptian records correct in recording Akhenaten to be the father of Ankhesenamun?

As for the KV21B mummy, the normal bones are in poor condition, but there is a poor quality skull and neck section available. If this skull has a good supply of teeth and the presence of the Lambdoid Cranial Suture [a joint between plates at the back of the head shaped like an inverted ‘y’], I would recommend using the accessible teeth and the Lambdoid plus any other suitable cranial suture to resolve her lifespan. I feel that this Lambdoid suture may hold the key to her age.

Currently we cannot identify KV21A or KV21B with any certainty. These two mummies could have been cousins, full or half-sisters, mother and daughter, mother and step daughter, or even grandmother and granddaughter. Perhaps, the age differentiation forensics and the complete DNA profiles for these two individuals could provide more information on the relationships of these two persons to each other and also to the Tutankhamun family members.

The York University team gave a good description of the dental condition for the KV35 boy and said that his proximal radius head was close to union but that all other bone ends investigated were not fused. In my opinion, this data gives a reasonable estimation of twelve to fifteen years at time of death. Both autosomal and mitochondrial DNA retesting should show if this mummy was the son of Amenhotep II or Amenhotep III, or someone else.

Conclusions

I propose that DNA retesting consisting of autosomal DNA, mitochondrial DNA, and amelogenin (the sex determiner) be performed on the fourteen mummies of the Tutankhamun family: of all eleven of the original Tutankhamun mummies and on the attainable mummies of the KV35 teenage boy, Amenhotep II, and Thutmose IV. The fragile nature of the two foetal mummies could give problems in yielding DNA. If full profiles plus mitochondrial DNA are obtained for all fourteen of these mummies, we should discover new and fascinating data on the strong interconnections within this family. Was there a strong possibility that Mutemwiya being related to Yuya or even Thuya? Was Amenhotep II the father of both Thutmose IV and Yuya? If this DNA examination is properly done, it will likely give us a more complete understanding of why the Eighteenth Dynasty died out, although a big stumbling block is the missing mummy of the pharaoh Ay. The use of age resolution forensics on the KV55 male, KV35 teenager male, and the two KV21 mummies could also help to reveal further information on the tangled relationships of Tutankhamun’s family.

Joseph L. Thimes
Dr. Thimes is a retired dentist with a particular interest in forensics, DNA, and human anatomy. He is a regular contributor to AE Magazine, including articles on fishing (AE121) and hunting (AE129).

Further Reading
Hawass, Z. et al (2010) “Ancestry and Pathology in King Tutankhamun’s Family.” JAMA 303(7), pp.638-647
Download pdf: https://jamanetwork.com/journals/jama/fullarticle/185393