A Forensic DNA Analysis of two Unusual Skulls of Uncertain Origin

By: Lloyd Pye

I. Forensic DNA Test Results On The Starchild Skull

The initial rounds of testing the Starchild's genomic (nuclear) DNA are now complete,
and the results are that it is male, and from a forensic standpoint it is human. There is
more to it than that, of course, because of the greater insights that might be available
through diagnostic testing, which is more extensive and expensive than we have been
able to afford up to this point. So what we will do now is go through the testing that
has been done, outlining the results, and after that we will discuss various views of what
the results mean and how they have been interpreted. I think you will find it fascinating.

The tests were conducted by Dr. David Sweet, Director of the Bureau of Legal
Dentistry (BOLD) at the University of British Columbia in Vancouver, Canada. BOLD
is one of the world's best forensic laboratories for the analysis and study of DNA
from calcified tissues, usually dealing with forensic cases less than 50 years old.
(The Starchild skulls have recently been dated at 900 years ago.) Dr. Sweet is an
odontologist (dental expert). During his work with the Starchild skull, he was
assisted by his Research Associate, Dr. Dean Hildebrand, a molecular biologist.
The academic credentials of both men are extensive and impressive, and their
professional reputations are impeccable.

They conducted a series of five genomic (nuclear) DNA tests: (1) one on the adult
skull found with the Starchild skull; (2) one on the piece of detached maxilla alleged
to be an integral part of the Starchild skull (see previous reports on this site for an
explanation of the detached piece of maxilla and its probable connection to the skull);
(3) one on a piece of bone from the Starchild skull known as an occipital condyle
(a piece of the foramen magnum, or neck hole opening); (4) one on the Starchild's
right mastoid bone (behind the ear); and (5) one a rectangular "window" cut from
the right-side parietal bone (the right side of the skull above the ear).

We will now briefly review each of those tests, first quoting relevant excerpts from the
official report, then expanding on that to make it clearer to nonprofessionals.

The first test (1) was on the adult skull, with results in on Nov. 2, 1999. Here is the
quote: "Human genomic DNA was extracted and typed from the adult's skull using a
screening test called AmpFISTR-Blue (3 genetic loci) plus Amelogenin (gender
determination). Results reveal that the DNA extracted from the adult skull (occipital
condyle) is from a female person. A genetic profile at three forensically significant loci
has been obtained."

This means everything went well and predictably with the adult skull. The condyle bone's
surface was sanded down to remove the usual traces of human contact, and the remainder
was chemically cleaned, crushed to powder, and prepared for testing. The Amelogenin
test, which indicates male or female, clearly showed female. Then a screening test of only
three genetic areas--the 3 loci (DNA sites)--was able to detect all of the DNA required
to provide a genetic profile of the female. This ease of extraction can be attributed to the
fact the human skull was not buried in the soil of the mine tunnel (or cave) it was found in.
It lay supine (face up) on the surface, meaning the condyle bone that was tested (a knob-
shape alongside the neck hole opening) would not have been touching the ground.

The second test (2) was run on the detached piece of maxilla (the upper right jawbone)
alleged to be part of the Starchild skull. That small piece of maxilla had two baby teeth
attached in an exposed position and three impacted in the bone above--two premolars
and one incisor. It was decided to test one of the exposed teeth because of ease of
duplication, ease of extraction, a high probability of recovering viable DNA (tooth pulp
encased in enamel is often the last part of a body to degrade), and the minimal damage
that would be done to the maxilla itself. Here is the test report on that procedure:

"Unfortunately, the results of PCR amplification of the sample recovered from the baby
tooth taken from the piece of right maxilla (upper jaw) was negative. No profile was
obtained. As I explained, this may be due to environmental factors, including humidity,
acidic soil, UV light, heat, etc. I think it will be valuable to harvest another small sample
from elsewhere on the exhibit and attempt another amplification procedure."

Polymerase Chain Reaction (PCR) amplification is how geneticists make infinitely small
pieces of DNA reproduce themselves into much larger samples that can be easily worked
with in a laboratory. However, when attempted on the crushed tooth extract,
there was no reaction! Thus, nothing at all could be said about the sex of its owner or
characteristics of its DNA, indicating that degradation of the sample was comprehensive.
This meant no genetic connection could be made between the maxilla and the
Starchild skull. Also, further testing on the maxilla was ruled out because of the poor
likelihood of results.

The third test (3) was done on the Starchild's occipital condyle, the same knob-like bone
from alongside the neck opening (foramen magnum) that had been taken from the adult.
Though smaller and less robust than the adult's, the Starchild's condyle was treated the
same in the lab. It was removed, its outer surface sanded away to remove all traces of
human contact (which would leave behind contaminating DNA), it was chemically
cleaned, then its inner core was ground to powder and tested. Here is the report:

"Human genomic DNA was extracted and typed from the child's skull using the same
screening test. Unfortunately, the DNA profile is a mixture of at least three people.
At the Amelogenin locus it was determined that at least one of the DNA contributors
was male (result is a mixture of male and female). This result indicates there has been
severe contamination of the specimen by DNA originating from several people. Despite
our best efforts to decontaminate the surface of the specimen prior to DNA extraction,
multiple DNA profiles were obtained."

This is self-explanatory, but a salient point not mentioned here that was discussed orally
is the Starchild skull's extraordinary porosity (a sponge-like quality) relative to normal
human bone. This might have been expected since the Starchild skull is remarkably light
compared to the adult skull, which is close to its size. (The adult was small, perhaps
only 5 feet tall, while then Starchild was large for a child, comparable to a normal
12-year-old.) Weighing only 1/2 as much as the adult, and perhaps 2/3 of a normal
six-year-old (the Starchild's presumed age), it is 1/3 larger, which has caused some experts
to question whether it is, in fact, a child. However, child or not, its bones are unexpectedly
durable.

We can assume half-weight adult bones would be thin and fragile, yet the Starchild's
skull bones, while indeed thinner than usual, show no cracking or fissuring apart from
normal (and extensive) cranial suturing. In contrast, the adult shows a severe concussion
in the upper part of her left parietal. If we accept that both lived and died in a rugged area
of northern Mexico around 900 years ago (calculated by a recently completed Carbon
14 dating test), we can presume the Starchild would have had ample opportunities to crack
a truly fragile head in the process of growing up in such an environment.

Others might argue that the Starchild could have been paralyzed, or virtually paralyzed, by its
aggregate deformities, and so would never be put in a position to be injured by the accidental
(or otherwise) blows to the head that all children are subject to while growing up. However,
others can argue that in such a place 900 years ago a paralyzed or otherwise helpless child
could not and would not have been kept alive because of the enormous strain that would place
on its family's undoubtedly meager resources. So it seems fair to suggest that extraordinarily
porous bones that somehow maintained reasonable durability are a sign of something beyond
the range of "normal" humanity.

The fourth test (4) was done on a sample taken from the Starchild's right mastoid bone
(mastoid process), the bone behind its right ear. The mastoid area is known to be porous,
too, but we were hoping to avoid cutting into the denser cranial bones and permanently marring
the beauty and symmetry of the skull. So extra grinding was done to the surface of the bone to
be certain to go into it below any possible seepage of oil from human hands that might have
handled the skull in the past. The result was then chemically cleaned, ground to powder, and
the test was conducted. Here is how the report reads:

"After the customary number of PCR cycles (28), there was a very weak gender profile
from the second bone sample taken from the child's skull (mastoid process). Other alleles
had 'dropped off,' which is usually a result of degradation of the genomic DNA. The approach
when this occurs involves reamplifying the sample with 5 additional cycles in an attempt to
produce a result. (The objective is to take whatever has amplified from the very tiny starting
amount of DNA and subject it to further amplification--in theory the DNA doubles with each
cycle so additional cycles may produce a result that can be visualized and interpreted.) Once
the additional 5 cycles were performed, which is the outer limit of current technology, only one
locus showed a profile: Amelogenin. The result is X-Y and this tells us two significant things.
First, the child was male; second, the DNA is human. Unfortunately, because we do not have
a profile from other loci it is not possible to conduct a paternity test against the genotype of the
adult skull."

This means that after a normal run of trying to amplify the DNA, it was found that degradation
(most likely from being buried in acidic soil, as mentioned earlier) had caused the alleles
(the actual gene segments that must be read for proper analysis) to "drop off," which means
they were reduced from long segments (strands) into bits and pieces so small they would render
almost no pertinent information. Try as they might, Drs. Sweet and Hildebrand could not amplify
those bits and pieces beyond sensitivity to Amelogenin (one of the least technically demanding
test procedures), which allowed them to determine its sex was male. This is a crucial
determination because it permits a conclusion by inference that the Starchild was human.
Here is how:

To obtain a sex determination of "male" means readings were obtained from both "X" and "Y"
chromosomes in the Starchild's DNA. From a genetic standpoint that means it received its X
chromosome(s) from a human mother and its Y chromosome(s) from a human father. >From a
forensic standpoint, even though virtually nothing else is known about the construction of the
Starchild's DNA, with X and Y chromosomes present, all of its finer details, if ever known,
would inevitably prove to be human. On the other hand, some might argue that without much
more clarity regarding the entirety of the Starchild's DNA, it is too early to rule out the possibility
that infinitesimally small fragments of other-than-human DNA might be present. Or, for those
willing to stretch a bit further, it could be that the biological template of the alien part of an alien-
human hybrid (which the Starchild might represent) would not be visible at all to any kind of
"human" testing.

These alternative views will be considered in more detail shortly. For now be sure to note that
the testing process could not establish a genetic link between the Starchild and the adult.
Consequently, we do not know if they were or were not a mother and her son.

The fifth test (5) was carried out to verify the "very weak gender profile" of the 4th test, and to try
to establish a genetic link between the adult and the Starchild. What follows is Dr. Sweet's report
regarding this final test (minus an introductory review of events up to that point, and a disposition
statement regarding repair and return of the skull), which was received by me on December 2, 1999.

I will enclose his words with the usual quotation marks, and wrap my explanatory comments in
brackets.

"Dear Lloyd:

After receiving from you permission to proceed, a section (4 cm x 4 cm) of parietal bone was harvested
from the lateral aspect of the right side of the child's skull. Rigorous decontamination procedures were
used to eliminate any contaminating DNA from the bone. Subsequently, 5.5 grams of powder were
produced from the sample by cryogenic grinding. The sample was divided in two and DNA was
extracted and purified from one half of the total amount. It was determined that there were 200
picograms of DNA present in this relatively large sample. (Ideal amount of target DNA for PCR is
1,000 picograms.) Thus, again, it appears that there has been considerable degradation of the DNA
over time due to environmental conditions at the site of discovery or during storage and/or transportation
of the exhibit."

[Since storage of the skulls is alleged to have been in cardboard boxes kept in garages for 50 to 60
years, burial in acidic soil seems the most likely source of a degrading influence. Also, by #5 all
testing procedures were being carried out with extraordinary rigor. And note that the recovered
sample is only 1/5 of what is needed for useful amplification.]

"Results-
PCR-based amplification of this trace of DNA produced an X-Y (male) profile but did not result
in supra-threshold results at other forensically significant loci. (No peaks.) The second half of the
sample was then processed and concentrated. PCR-based amplification of the DNA produced
the same result as with the first half. That is, X-Y (male) and no peaks at other genetic loci
tested."

[Amelogenin gave another X-Y read as a male, which allows the extrapolation that both the X
and Y chromosomes had to come from humans, as occurred in test #4. And there were still no
significant (supra-threshold) results generated, which means not enough DNA was recovered to
complete allele calls that would allow paternity testing.]
 

"Conclusions-

Due to the strict cleaning regimen employed with this sample, it is my opinion that the DNA that
was isolated and tested was not from exogenous, contaminating DNA. The result appears to be
due to the age of the skull; the genomic DNA is too degraded to provide a complete profile. The
sex of the decedent has been verified as male. The traces of DNA that were recovered in each of
the numerous tests performed in this laboratory responded to human-specific probes."

[Human-specific probes are segments of DNA which respond only to complimentary sequences
of base pairs in human DNA at flanking regions of the locus (site) under study, (which in forensic
testing is normally 100 to 300 base pairs long). Thus, they will not match up with anything other
than counterparts within human DNA. This means there is a certain detectable amount of human
DNA in the Starchild. It does not guarantee there is only human DNA present, nor does it indicate
there is anything other than human DNA present. In other words, human-specific probes are
indicative but cannot be definitive. They can imply or suggest innate humanity, but they cannot
prove it beyond doubt.]
 

"Recommendation-

The following question arises: Can DNA be used to evaluate, assess, or diagnose the etiology
(cause) of the unusual shape and appearance of the child's skull? Unfortunately, this laboratory
deals only with STR loci that have forensic significance -- they are non-diagnostic loci. The
specimen would have to be tested by a laboratory that focuses on diagnostic genetic loci if one
was to consider attempting to identify a potential genetic cause for the unusual appearance.
Further, it is predicted that diagnostic laboratory will also find the same difficulty in isolating
and extracting sufficient DNA for genetic testing."

[This is the crux of the matter. What we have now are test results for "loci that have forensic
significance." The forensic testing of calcified tissue (bone) is done with markers in the range of
100 to 300 base pair lengths, which is adequate for answering broad-based questions like,
"Is a sample male or female? Is one sample related to another?" Or even, "Is it human or ape
or cow?" Such determinations can be critical in situations where old bones are recovered in
an isolated, unmarked grave and foul play may be suspected. On the other hand, diagnostic
testing is done with markers longer than 500 base pairs in length, which provides a much
finer determination of DNA characteristics.

What we must decide now is whether to move forward with diagnostic testing on the Starchild,
which could tell us whether or not its physical anomalies are due to some kind of known
chromosomal disorder (Down's syndrome, hydrocephaly, etc.). That potential gain must be
weighed against the assured cost, which will be much greater than forensic testing (and is a
primary reason we opted for forensic testing in the first place). Also, any diagnostic lab will
face the same degree of degradation the BOLD lab encountered.]

"Sincerely,

David Sweet

Director, BOLD Lab"
 

In closing this part of the website update, I would like to compliment Dr. Sweet and Dr.
Hildebrand for professional diligence above and beyond the call of duty. We paid for the
first three tests only, with the cost of the next two coming out of their pockets because
they wanted to secure the best possible results they had contracted to deliver. Nowadays
we seldom find that kind of commitment to the old-fashioned ideal of value for money,
and we want our gratitude for their integrity and competence to be formally recognized.