Tracking RNA to Pinpoint Time of Death: Better Than Bugs?

DNA is a persistent molecule. Genome sequencing is possible for creatures as ancient as mummies and mammoths. But the messenger RNA (mRNA) molecules that translate a gene’s information into a specific protein are more ephemeral, waxing and waning in a tissue over time, even after death, due to the instability of the sugar part of the molecule.

A multinational team has adapted the changeability of gene expression – mRNA production – into a computational tool that uses transcriptomes – the sets of mRNAs in particular body parts – to deduce time since death. In forensics terms, that’s the postmortem interval (PMI). Roderic Guigó led the effort that includes researchers from Portugal, Spain, and the Broad Institute at Harvard. Their report appears in Nature Communications.

The Worms Crawl In, The Worms Crawl Out

Classic measures of the PMI include algor (change in body temperature, not a former vice president), livor (blueness), and rigor mortis (stiffness). More quantitative is forensic entomology, which deduces the PMI from the developmental stages of insects as they consume a corpse. The residents are mostly the larvae of blow flies, flesh flies, and the occasional beetle.

It’s an old and obvious way to assess time since death, such as looking at the squiggling white wormy things in the rodent heads that my cats bring home. “Use of forensic entomology is recorded as early as the mid-1300s, during a murder investigation in China. In 1855, a French physician named Bergeret determined that the insects in and around a baby’s corpse, found behind the plaster mantle in a house, placed the time of death back several years, thus implicating the former, and not current, homeowners,” I wrote in “Where the Bugs Are: Forensic Entomology” in The Scientist.

I remember from my years of intimate acquaintance with fruit flies that distinguishing the larval stages can be difficult, requiring familiarity with mouth parts, breathing tubes, genitalia, various hairs and other protuberances, and abdominal pigment patterns. I only used Drosophila; the munching maggots of a murder scene form a diverse community.

In 1994 the field began to include single gene sequences to identify species, but still a flummoxed forensic entomologist might have had to rear a mysterious larva to adulthood, on beef liver, to see what it was! But we’ve come a long way. In 2016, the common North American blow fly, aka the “forensic timekeeper fly,” had it’s genome sequenced. These flies are among the first to move onto and into a human corpse, within minutes of death. When the females lay eggs, the developmental “clock” that forensic investigators follow begins, from which they deduce the postmortem interval.

A DNA approach is also useful to probe the contents of insects’ guts for further clues to the crime, although their intestines degrade human DNA in just 2 days.

And now the evolution of forensic entomology has evolved from insect parts and DNA to the RNA of the victim.

Changing RNA Levels After Death

Crucial to the new work is the “Genotype-Tissue Expression Project” portal at the Broad Institute, aka the GTEx tissue bank. It compiles RNA sequences from human tissues (fat, muscle, blood), organs (stomach, lung, heart), and body parts (lower leg).

The researchers first looked at RNA in thousands of samples – including spleen, esophagus, small intestine, ovary, prostate, heart, nerves, salivary glands, and skin with and without sun exposure – from 540 donors. They identified the genes that were transcribed into messenger RNAs whose abundance changed, up or down, as the body cooled after death (making mRNAs is also called gene expression).

Body parts die at different rates and in different patterns. In muscle the expression of many genes changes fast; in the liver changes are slow and steady, which makes sense considering what these parts do. In general, though, RNA patterns stay true to tissue – the genes active in a living liver are the ones cranking out mRNAs in a liver shortly after death, and these differ from the genes active in a nerve or a bone.

The investigators focused on blood, because it not only holds clues in its easily-measured components, but blood draws taken before death could extend the timeline back in the analysis to a known point.

DNA is still readily transcribed into RNA after cardiac and brain death. For the first 7 hours many genes are expressed more than they were before death. The 7-to-14 hour interval shows the most fluctuation, with thousands of genes being transcribed much more or much less than they were when the person was alive, as if the system of DNA-to-RNA-to protein is going haywire as it shuts down. From 14 through 24 hours after death, some genes are still being transcribed at a faster or slower rate than they were when the person was alive.

A New View of Death

The waves of changing gene expression paint a panorama of death at the microscopic level.

Cell division stops.

The white blood cell count, especially neutrophils, plummets, perhaps because infection is no longer a threat.

Necrosis (cell death from injury) ensues, while blood clotting, DNA repair, and the stress response increase.

Ion transport, carbohydrate metabolism, and cholesterol synthesis halt.

Cells shift from aerobic respiration to the anaerobic route, as the lungs no longer bring in oxygen nor does the circulation deliver it.

The timeline of death revealed in the patterns of gene expression is more than the reverse of the “characteristics of life” list that begins most introductory biology textbooks. It’s useful.

The researchers used their data to create prediction software that can tell the time elapsed since organismal death from the RNAs in just four of the 36 tissues studied: subcutaneous fat, lung, thyroid, and sun-exposed skin on the lower leg. When they tested the software on 129 bodies with time of death known but blinded, the results were accurate to within 10 minutes of the actual time of demise.

Beyond Forensics to Transplantation

Tracking RNA to determine the PMI may make forensic entomology less messy and more precise. The researchers point out another potential application: identifying tissues and organs viable enough for transplant.

Most organs are harvested after complete brain death in a hospital, but increasingly organs are being donated after the heart stops and some brain function remains, but is so minimal that chance of recovery is nil. In the first case, the organs don’t cool after circulation stops – they’re removed and prepared right away. But in the second situation, “donation after cardiac death,” hours of cooling may pass, similar to the “cold ischemic interval” in a murder case.

The RNA test can perhaps determine which tissues and organs haven’t gone too long without oxygen, and are transplantable. And the time is short. A heart lasts 4 hours, a lung 4 to 6 hours, a liver 6 to 10 hours, intestines 6 to 12 hours, a pancreas 12 to 18, and kidneys 24. Organs procured after full brain death in a hospital have a 24-hour window. Might RNA profiling extend sources of organs beyond hospital deaths?

Although organ donations hit an all time high in 2017, a spike unfortunately partly due to the opioid crisis, 116,000 people are now on waiting lists in the U.S. for transplants. Perhaps the new technique can be used to increase the numbers of available body parts: hearts, livers, kidneys, lungs, pancreases, small intestines, corneas, skin, veins, heart valves, tendons, ligaments, and bones.