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DNA in the Trenches: How to Identify Fallen Soldiers

Can DNA survive 100 years and lead to the identification of a WWI soldier? Yes, but not the DNA you’re thinking of.

Last week, we were told that DNA performed a bit of a miracle: it identified a man who was born in 1894 and who died during the First World War. His name was Percy Howarth. He was born in Lancashire, England, and moved to Canada a few years before enlisting with the Canadian Expeditionary Force. In 1917, he fought at the Battle of Hill 70 near Lens, France, as part of the 7th Canadian Infantry Battalion.

He never came home from that battle and was presumed dead at the age of 23.

Over a century later, his remains were discovered during a munitions clearing process in France alongside those of four other Canadian soldiers, which were quickly identified. But the identity of Howarth’s remains could not be pinned down for years.

Soldiers do not always return from war. Focusing on the two World Wars and the United Nations Operations in Korea, Canada’s Casualty Identification Program reports that we have over 27,000 Canadian war dead with no known grave. South of our border, it’s more than 72,000 Americans who are unaccounted for during World War II alone.

I can’t speak to the exact process by which Corporal Percy Howarth was identified. However, having worked for the Armed Forces DNA Identification Laboratory in the United States, a facility which is part of a similar process of identifying the remains of prisoners of war and personnel missing in action, I know how they went about using DNA to figure out whose bones were found in France.

It may seem like an impossibility. How could DNA have survived the elements for a hundred years?

Mother knows best

DNA is present in the nucleus of most cells. For a mental picture, imagine cracking an egg onto a frying pan. The result looks a lot like a cell, with the yolk being the nucleus. Inside that nucleus is a lengthy DNA molecule that has a few limitations when it comes to surviving death. Every bit of information on it is only present in two copies, and each copy can be slightly different since one comes from your mother and the other from your father. Given this low number of copies, DNA found in the nucleus rapidly degrades beyond recognition after death. Molecular scissors known as nucleases are released from inside cells and start chopping up the DNA. Bacteria, fungi, and insects have a field day with the remains. Ultraviolet radiation from the sun, meanwhile, introduces bends and kinks in the DNA molecule which will cause problems for scientists down the road. If DNA is so easily damaged, how can it be used to identify a World-War-I-era soldier?

The twist is that this DNA, called nuclear DNA after the word “nucleus,” is not the only DNA inside our cells. The DNA mentioned on television shows and in books is almost always nuclear DNA and the chromosomes that it forms, but in the “egg white” of our cells (which is called the cytoplasm), we have tiny structures, mitochondria, and inside these energy-generating mitochondria lies a different kind of DNA.

For the purposes of human identification, mitochondria are fantastic for two reasons. First, each mitochondrion contains DNA, and each cell contains hundreds to thousands of mitochondria. This means that if one mitochondrial genome is mauled beyond recognition by UV light, moisture and heat, there are hundreds more copies available for testing.

Another advantage of the mitochondrial genome is that it is circular. While nuclear DNA is a linear molecule, the mitochondrial DNA molecule is shaped like a ring, which makes it hard for molecular scissors, which often need a clean end to latch onto, to chew it up. With such strong benefits for mitochondrial DNA, we might ask, “Why even bother testing nuclear DNA found in crime scenes?”

First, the size of the usable portion of mitochondrial DNA for human identification is quite small. The longer the DNA molecule, the more bases can mutate, the more variability is generated, the more discriminating this DNA molecule becomes. A short DNA sequence has a limited power of discrimination, meaning the ability to tell two people apart by looking at their DNA.

Second, while its mutation rate is considered by scientists to be relatively high (much higher than that of the nuclear DNA used in contemporary forensic cases), it is not nearly as high as you may think. Different studies calculating this rate using varied methodologies report one mutation every 33 generations or one every 233 generations. This means that the mitochondrial genome is generally transmitted from one generation to the next as is, which makes discriminating between individuals a bit harder.

Third, a mechanism called homologous recombination, which generates a lot of genetic variability during the production of sperm and egg cells, is traditionally thought not to occur within mitochondrial DNA in humans. This limits the creation of interesting variation. The reason why homologous recombination, which affects nuclear DNA, is not known to occur in mitochondrial DNA is that the latter is transmitted to children in a peculiar way.

You received your mitochondrial DNA exclusively from your mom. She and all of her siblings, regardless of sex, got it from their mom, and so forth. This is called “matrilineal descent.” The reason mitochondrial DNA never comes from the father is that the mother’s egg supplies most of the cytoplasmic material (the “egg white”) for the embryo. Since the mitochondria are in the cytoplasm, none are passed on by the father. (Some scientists recently claimed this dogma had been upended, but this was not the case.)

At the mitochondrial level, you are identical to your siblings and quite probably to your mother and her siblings… and their mother and her siblings… going back dozens of generations. If crime scene analysts were to exclusively use mitochondrial DNA for identification, you could be convicted of a murder your brother committed.

However, if no nuclear DNA has survived the environmental conditions of a French mass grave for a hundred years, mitochondrial DNA can become a useful piece of the human identification puzzle.

The military-mitochondrial complex

The United States government claims to have the largest forensic skeletal laboratory in the world, and if you’re going to have the largest forensic skeletal lab in the world, you might as well build it in paradise.

Hawai’i.

That’s where the Defense POW/MIA Accounting Agency laboratory is located. Its mission is to search for, recover, and identify U.S. personnel missing from past military conflicts. To this end, historians and military analysts research individual missing-in-action cases by interviewing foreign and American witnesses, by collecting information from veterans’ organizations, and by sifting through U.S. military records and local newspapers. Once a likely site is identified where the remains might be found, a field investigation team is deployed to that location to assess the likelihood of the prediction and the feasibility of an excavation. When given the green light, a recovery team of 10 to 14 people, including a forensic anthropologist, is flown to the site where it will spend 35 to 60 days excavating, calling upon the locals to help in the effort.

When bone fragments are found, they are sent to the laboratory in Hawai’i where anthropologists try to reconstitute useful information from those bones: sex, age, stature, and any trauma or illness that may be shown by the osteological evidence. Similarly, if jaws or individual teeth are found, forensic odontologists examine the evidence and compare their findings to military dental records. In 70% of cases, however, anthropological and dental evidence is not enough: that’s where DNA comes in.

Under sterile conditions, a piece of the recovered bone fragment is sawed off and shipped to a molecular laboratory known as the Armed Forces DNA Identification Laboratory or AFDIL. Once it has been properly documented, its mitochondrial DNA is extracted and the sample is ready for PCR amplification. This is the same basic technique that is used to test for the presence of the coronavirus in COVID-19 laboratory tests: it involves copying parts of the DNA molecule many times over, in much the same way that a single page from a book can be photocopied over and over.

The mitochondrial DNA molecule contains a few genes, some of which can be mutated and lead to diseases. These genes are never amplified in the course of forensic work, as they contain potential medical information which would result in sticky ethical issues if were they unearthed during human identification. Rather, forensic scientists look at the control region of mitochondrial DNA, which does not contain genes but rather a lot of useful variation.

Once that variation has been documented and a profile has been established, it needs to be compared to a reference sample. Unfortunately, the U.S. Armed Forces only started collecting DNA on its service personnel in 1992. For most American military personnel who went missing in action in conflicts older than the Gulf War, no DNA is directly available from military records… but it is available by proxy.

Because mitochondrial DNA goes down the matrilineage mostly unchanged, any relative of a missing service member sharing that lineage can contribute their mitochondrial DNA as a reference. Indeed, service casualty officers regularly collect skin cells from the mouths of maternal relatives of personnel that is missing in action, and these skin cells are sent to AFDIL for extraction and sequencing. A DNA match, however, is not inherently proof of identity, as more than one person can share this profile, so it becomes one piece of the puzzle which must be completed by the location of the find, details from any wreckage, found objects, pieces of cloth, and witness testimonies.

In the case of Corporal Percy Howarth, a number of clues were helpful in narrowing down the search. The shoulder of his uniform indicated he had been fighting with the Canadian forces. A whistle found with him suggested his rank was above that of a private. The age and height of the remains whittled the list down to a few dozen names.

Mitochondrial DNA could be extracted but it needed to be compared to that of a living maternal relative and tracking down one of Howarth’s was not easy. Eventually, the phone rang at Carolyn Cooling’s house. Ms. Cooling is Howarth’s great-great niece. Thanks to her DNA donation, the Canadian Armed Forces Casualty Identification Program was able to get a match.

The next time you hear about DNA being useful to identify someone, you’ll know to ask, “nuclear or mitochondrial DNA?”

Take-home message:
- The remains of Corporal Percy Howarth, who died during the First World War, were recently identified using, in part, mitochondrial DNA testing
- Mitochondrial DNA is a small, ring-shaped genome found in the energy factories of our cells
- It can be used to identify very old remains that have been left out in the elements because cells contain hundreds or thousands of copies of it and because its circular shape helps protect it from degradation by enzymes


@CrackedScience

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