DNA as an Eyewitness

How Phenotyping is Being Used to Predict a Suspect’s Appearance

Ann MacPhetridge, Promega

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Like many teenage girls in the 1990s, Dr. Susan Walsh was fascinated by Agent Dana Scully’s work as a forensic pathologist on the television series, The X-Files. Already interested in understanding how humans work and function, watching Agent Scully use science to try to solve highly irregular cases helped to inspire Susan to pursue a degree in Biochemistry from University College in Cork, Ireland , followed by a Master’s degree in Forensic DNA Profiling at the University of Central Lancashire.

She further pursued a PhD in Forensic Genetics from Erasmus University and came to the US to do a post-doc at Yale University.

“… When I was ready to do a PhD, I thought long and hard. And that’s what I say to students as well, “Don’t just jump into a PhD, this is 4 to 5 years of your life, so you’ve got to hold back and really, really make sure that the topic that you’re picking is what you want to do. I literally just Googled ‘appearance forensics DNA’ and up popped Dr. Manfred Kayser. I sent him an email and the rest is history, really.”

After a post-doc at Yale University, she applied for a post for a combined biology and forensics professorship at the Indiana University and Purdue University – Indianapolis and has been there since August of 2014.

Recognizing that there was much to be gained in the field of forensics from DNA phenotyping, she wanted to research methodologies that would enable more information to be gained from the DNA taken from crime scene samples. Dr. Walsh’s earlier work was focused on using SNPs to enhance understanding of the genetics behind determination of human physical appearance and ancestry. This information can be used in a forensic casework context to provide key intelligence information such as eye, hair or skin color to law enforcement. While this information is not comprehensive, it can be used to give direction to law enforcement as an investigative lead in an otherwise tough missing persons case or in a mass disaster situation.

“As we deal with quantitative traits where different genes and also environmental factors can contribute to the phenotype, we try to unveil the genetic basis of certain traits using genome-wide association approaches and next-generation sequencing, amongst others. We then try to determine what are the most-predictive biomarkers and develop molecular tools to predict these traits for practical applications in forensics and anthropological studies.”

An Overview of the DNA Phenotyping Process

The forensic DNA phenotyping workflow is not unlike that of laboratories performing now on their casework samples. Laboratories follow the same steps and utilize the equivalent types of instruments they would be using if doing autosomal STR analysis: the crime scene sample is collected, DNA is extracted and quantified from that sample, and then a STR profile is generated. The results that laboratories obtain from that profile can then be used to determine next steps. In the case where there is no comparable hit to a database, then intelligence gathering methods such as DNA phenotyping may be a valuable option. Forensic DNA phenotyping requires minute amounts of DNA. In fact, the typical input amounts of 500pg to 1ng of DNA used in STR analysis on CE instruments are much greater than what is required to perform DNA phenotyping. While there are additional clean up steps and an extra PCR step or two, the workflow is generally the same. “We look at peaks that are similar in color to that of STRs and where instead a nucleotide is shown, we have an A, C, T, and G to show the variants that we need.”

Massively parallel sequencing (MPS) has revolutionized DNA phenotyping. Capillary electrophoresis limited the number of SNPs that could be used in assays. With MPS, laboratories can combine hundreds to thousands of SNPs. “We have the freedom now to say that although it’s only introducing a 0.05% increase in prediction accuracy, just do it because it’s not impacting our assay design. This gives us a tremendous amount of freedom when designing prediction models because we can include minor contributors, interactions, and it helps push our research. We’ll see more and more accurate prediction models made because we’re able to put in 100s more variants than before.”

Another Tool in the Toolbox

The accuracy of forensic DNA phenotyping has improved tremendously in recent years. A great deal of the accuracy differences between assays is related to how continuous the trait is in general. For instance, there are fewer categories associated with eye color than with hair color or skin color, so eye color is generally easier to predict. Results are often reported in categories, not with continuous color, because continuous color is more difficult to predict despite having a more accurate phenotype.

Pigmentation has approximately an 80% accuracy rate, with a lot of the errors being demonstrated in the boundaries of the categories. The correct variants are known but understanding the key combinations of color in various populations is still being studied, with more work to be done. Various programs exist as tools for hair structure, male pattern baldness, and height.

“Height, although it has been studied the longest, is also one of the most difficult traits. You need approximately 700 variants to get at least 70% accuracy. And even at that you are talking millimeters to centimeters differences for the phenotype. We can get the extremes--very tall or very small, but at the end of the day you are investing, many, many markers for something that I don’t think we’re going to significantly improve on. The key with phenotyping is to know where the wins are. Recognizing that we won’t be able to understand the environmental impact on many of the appearance traits.”

"The key with phenotyping is to know where the wins are. Recognizing that we won’t be able to understand the environmental impact on many of the appearance traits.”

Her lab has worked with law enforcement, but few details have been shared by those agencies on the conclusion of the cases. Engaging with practitioners at conferences such as the International Symposium on Human Identification enables her to discuss practitioner needs in more detail with laboratory staff. Hearing firsthand about what modifications to a workflow would be acceptable versus not helps direct the assays she and her team are developing.

In a situation where a heinous murder or sexual assault has been completed and an active investigation is underway, a laboratory is often pushed to turn around their casework results as soon as possible. What many laboratories may not know is that a phenotype can be generated in the same amount of time as a DNA profile. Since DNA phenotyping requires very little DNA for the assay, running an STR assay concurrently with a phenotypic assay could provide relevant, actionable information, particularly if conventional DNA profiling is not that useful.

Genetic genealogy, a method which has seen a massive increase in interest since the arrest of the Golden State Killer in April of 2018, could also benefit from DNA phenotyping. Genetic genealogy often gives you hundreds of scenarios that the investigative team can pursue. By utilizing the phenotypic results, detectives could narrow down the number of potential suspects. For example, if the phenotypic result predicts that the DNA comes from an individual with blue eyes, investigators could concentrate on families where blue eyes are more prevalent to find that connection.

To those readers who may be concerned about privacy violations, Dr. Walsh reminds them that DNA phenotyping is very good at predicting with broad groups and does not produce individualized results. In the scenario of a case, investigators can then narrow their focus based on these broad group predictions. At the end of the day, it remains nothing more than an investigative lead. She feels that it’s actually less intrusive than other techniques and tools used by law enforcement such as CCTV.

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Hurdles for Wider Adoption

The lack of numerous validated commercial kits is limiting the adoption of this methodology. Incorporating kits and protocols developed in a research laboratory is not a viable option for most casework laboratories who simply lack the expertise and required resources to optimize a kit. Instead, mass produced kits that have undergone robust manufacturing and quality control processes are needed for DNA phenotyping to be more widely adopted by the community.

In addition to the lack of available kits, bioinformatics continues to be a hurdle. Laboratories often lack bioinformatics experts. The vast amounts of data that is generated by MPS is daunting to laboratories who are often juggling backlogs, staffing shortages, training challenges, and ever shortening deadlines. For analysts to feel comfortable testifying in court, there must be a clear understanding of the algorithms used to analyze the data. This understanding must be balanced with the need to also make kits that are user friendly and easy to troubleshoot.

"What many laboratories may not know is that a phenotype can be generated in the same amount of time as a DNA profile. Since DNA phenotyping requires very little DNA for the assay, running an STR assay concurrently with a phenotypic assay could provide relevant, actionable information, particularly if conventional DNA profiling is not that useful."

From Eye Color to Facial Morphology

Facial morphology is a generally unknown research area with tremendous potential in terms of prediction. Still in its early stages, the current work is devoted to understanding the genetics and how genes contribute to pathways. Dr. Walsh pointed out that this basic groundwork for eye color and pigmentation was done more than a decade ago. It’s essential to understand what the genes are doing, how they are contributing to a pathway, and how are they may be different between populations. Forensics and biomedical teams are learning from one another’s research and subsequently applying that knowledge to their own research, resulting in advances that mutually benefit both groups’ work. Understanding what is contributing to bone structure and cartilage is one such area where both groups are focusing their research energies.

Through collaborating with prominent researchers such as Dr. Manfred Kayser (Erasmus University), Dr. Peter Claes (KU Leuven) and Dr. Mark Shriver (Penn State University), much of the research performed by Dr. Walsh and her team is on genome wide studies and proving which genes are essential for prediction versus not, with the goal of determining which variants are causal variants and are responsible for contributing to pigmentation, and facial morphology.

Much of the work on facial morphology is centered on dividing the face into several regions and analyzing what variation exists between individuals in that particular area of the face.

“An interesting aspect of the face is that it is not flat. The face is a 3D structure. Trying to predict not only one dimension, but trying to predict several dimensions. The work of Dr. Peter Claes is really revolutionary. He is able to take his engineering research and apply it to the face. Peter is taking a data driven approach to understand the face. He uses the face’s variation of multiple individuals to tell us what structure exists within the face. From there we try to understand what a gene may be doing in this part of the face…What kind of variation exists between individuals…it’s so exciting”

While the technology allows for predicting the curve of a lip or the structure of a chin, predicting the sides of one’s face is nearly impossible. Body mass index, age or other environmental factors contribute to the fat tissue that is deposited on the bone and influence the makeup of the sides of the face. And once it becomes possible to predict a complete facial appearance, it will not be feasible to predict one specific face and its various features. Because of the variations of environment and diet, the programs will be required to produce several faces for identification purposes. “It will be up to the multiple faces that are predicted to give an impression of what a face could look like by combining the features of several faces. But we’re still a long way from that. Let’s find the genes and then we can see what we can do next.”

DNA Forensics in 2025

Dr. Walsh predicts that genetic genealogy practices will be shifted to many laboratories. The successes seen in numerous cold cases has opened the door for investigators to want more resources devoted to this type of work. Because of the expertise required to do the ancestry analysis such as family trees, she feels that standards will need to be put in place first. Standardization has benefitted so many other areas of forensics work, it is not unreasonable to anticipate more standards and guidelines put into place before it is more commonly used.

She also believes that tools that help provide insight into appearance will continue to be developed and mainstreamed. Inferring ancestry can go alongside phenotyping and the research can benefit both areas. Everyone is interested in appearance. Ancestry inference and microhaplotypes will become more important to human identification and goes hand in hand with DNA phenotyping.

Another area she feels passionate about is methylation and the insight that it can provide to inconsistencies between phenotypes and genotypes.

“Methylation fascinates me. Unanswered questions as to why we see particular traits or phenotypes when genotypes don’t dictate that. Methylation is key to understanding that. Anyone studying methylation is in a hot spot, but there are some current limitations due to treating the DNA with bisulfite treatment for conversion and then going off to do sequencing. So much to do, so little time.”

Leading the Way

Like many women in our field, she believes that the forensics field is leading the way in regard to identifying female scientists as leaders and giving them opportunities in a variety of roles to lead teams. In her case, Dr. Manfred Kayser acted as a great mentor and has been extremely supportive of her research and sees her as an equal and has shown a sincere appreciation for her contributions to the field and to their research.

“If you have a goal, if you have drive, this is the area where we are seen as leaders. We’re making a difference. Over time, we will see more female researchers in panels and on editorial boards. Forensic science is leading the way. We are pushing for equal numbers and having women in higher positions. Think of Mecki Prinz…she was one of the leaders and she’s been at the top of ISFG pushing female researchers to go and go and go.”

Her advice to young women interested in a career in forensics, echoes the same advice she was given years ago by her biology teacher. “Don’t focus just yet. Get a broad degree so you can see the bigger picture and then you can apply it. Bring the broad exposure of your undergraduate degree to forensics. Wait until you are absolutely sure for a PhD…You should have passion from Day One. You must feel like you want to be the one who goes in to solve it, because you want to figure it out.”

Juggling Motherhood and Research in a Pandemic

Not surprisingly, the COVID-19 pandemic changed how she works. As a mother to two young boys-aged 3 and 5 years old-she found it difficult to be both mom and a scientist. The amount of work that she’s able to accomplish now isn’t at the same level as it was pre-pandemic.

“It’s difficult to do both. You just can’t do both. My work did suffer in the past few months. Students have been great; they’re still working from home, thankfully we haven’t stalled to nothing. I haven’t been at the level of research I was at before this all began. The positive side of this is that you realize there are times where you just have to optimize your time more efficiently, be very focused, and take a ‘I’ve got to get this done’ approach.”

So Susan’s adapted by applying those small amounts of time to her advantage. If she has an hour to spare, she manages it to the best of her ability. When asked what superpower she would like to have, it was no surprise to hear her say, time management. Being able to control time, to slow it down a bit to get more done would be ideal right now. I think many of us can agree with that sentiment.

While it may seem to the lay person to be something out of a science fiction movie, the work on DNA phenotyping continues to advance the field of forensics in new and exciting ways. Being able to accurately predict facial and body features may help investigators by focusing their search for an unknown perpetrator who may not be identifiable with standard STR analyses.

Dr. Walsh and her team embrace the opportunity to work with law enforcement and forensic casework laboratories across the globe. If you have a case that you feel may benefit from their DNA phenotyping research, email Susan at walshsus@iupui.edu. To learn more about her work and the work of her team, visit the laboratory’s website.

Dr. Walsh will be presenting "Quantitative Prediction of Eye Color from DNA" at the 31st International Symposium on Human Identification this September.

Dr. Susan Walsh

Dr. Walsh is an Associate Professor at the Department of Biology and Forensic and Investigative Sciences at Indiana University Purdue University Indianapolis. Dr. Walsh completed her PhD in Forensic Genetics at Erasmus University in the Netherlands. She went on to complete post-doctorate work at Yale University before joining IUPUI in 2014. She is the proud mother of 2 young boys and is grateful to her husband who enables her to focus on her research. “I couldn’t have all this (my own lab/research) without my wonderful, supporting husband. During this Covid-19 pandemic we have certainly acted like a tag-team AGAINST our kids hahaha!”