Elucidating the Blood Group Regulome

What have your Ph.D. studies focused on?

“Blood transfusions are used to help patients experiencing a wide range of health conditions from anemia to complications during childbirth. Globally, each year, nearly 120 million units of blood are donated to be used in these situations. Although the safety for both donors and blood recipients is significantly improved, when it comes to blood transfusions there are still some risks attached, especially regarding reactions due to incompatible blood types.

On the surface of our red blood cells are structures, known as antigens, which can trigger our immune system to produce antibodies. Across the human population, there are currently 390 antigens recognized and we can categorize the majority of these antigens into 45 known blood group systems, two of which you might be familiar with – the ABO blood group and the RhD group. The antigens that make up these two blood groups are what determine a person’s blood type. Blood types are commonly known as types: A, B, AB, or O followed by positive or negative sign, which indicates whether a person has the RhD protein or not.

And while the types of antigens can differ between people, the amount of one antigen an individual has can also differ from person to person. The concern here is if a minimal amount of an antigen is present on red blood cells, this could potentially lead to challenges in blood testing during patient and donor matching. For instance, being identified as type O (negative for both A and B antigen), when you are a weak B positive, can complicate blood transfusions. Our idea was that this issue of actively weak antigens – by that I mean an individual has a lower number of antigens – could be down to a gene regulation problem. So, this is what my thesis is centered around.

When the gene is regulated by binding transcription factors - proteins that regulate how fast genetic information is copied from DNA to messenger RNA – it can boost the production of transcription meaning that you have a lot of mRNA and a lot of protein. This also means that you display a lot of antigen. However, if you lose the binding of transcription factors, for example, due to a genetic alteration, then you do not get this boost, meaning that your antigens could be minimally expressed or even not expressed at all. There are known examples of this in blood groups, like the Duffy blood group, that a single mutation in the promoter region disrupts transcription factor binding and abolishes the expression of the protein completely. 

We had this concept in mind as we set out to find more of these mutated transcription factors’ binding sites. But instead of having any predetermined questions, we decided to look globally at what we could find across all the blood group genes. In fact, our hypothesis was that quantitative blood group variation depends on differences in binding of transcription factors, at least partially.

Taking a systematic approach, we looked at all the blood group genes and for each, probed the question: What alters the expression levels of this gene? To study blood group gene regulation and where these transcription factors were binding in the DNA, we had to purposely develop a bioinformatics pipeline.

With the pipeline in place, we were able to identify the mechanism behind a very weakly expressed CR1 (complement receptor 1) blood group called the Helgeson phenotype, which is detailed in my first thesis paper published in Nature Communications. Researchers have known about this antigen and the weak phenotype since 1970 but have not been able to identify the mechanisms behind it. It was known that this antigen had something to do with the immune response and was associated with malaria, as CR1 is a known receptor for the parasite. It was also known that CR1 could be very weakly expressed on the surface of red blood cells.

What we found was that a gene variant in intron 4 of the CR1 gene is what causes the very weak expression of CR1 on the surface of red blood cells, interestingly this does not affect CR1 expression on immune cells or other tissues. After we learned this, we expanded our pipeline starting first with GATA1, another well-known transcription factor, before moving on to investigate other transcription factors, including several that are important for red blood cells.

We began thinking that epigenetics could also play a role, as open chromatin regulation is needed for a transcription factor to bind to the DNA. So, we incorporated ATAC-seq and histone modifications, indicating promoters and enhancers, where transcription factors bind. We again extended our pipeline to help us be more precise in our predictions, and this process is highlighted in my second paper.

Then lastly, in my third paper, we applied this upgraded bioinformatics pipeline to clinical samples. Here, we looked at the RhD, which also needs to be matched for blood transfusions. We analyzed 13 blood samples that we received from the blood bank here in Lund, and from our collaborators in Australia, using the same systematic approach to find where the transcription factors bind and whether that affects antigen expression. In one sample we found a novel variant in the promoter of RhD that again disrupted the binding of GATA1, one of the erythroid master regulators. So, we were able to use this pipeline in more translational research using actual clinical samples, which shows the purpose of my thesis, which is to ensure the safety of blood transfusion,” explains Gloria Wu.

How did you end up doing a Ph.D. at Lund Stem Cell Center?

“I was raised in Taiwan and have a degree as a medical technologist (MT), which is equivalent to a biomedical analyst (BMA) degree in Sweden. Following my university years, I worked at a blood donation center in Taiwan. While there, I became fascinated by blood groups, the varying blood types of individuals, and the importance of matching patient and donor blood types for safe transfusions. This curiosity led me to explore sequencing, particularly next-generation sequencing, with a focus on genotyping blood groups to explain aberrant phenotypes thus to streamline the matching process with a single test. The blood center served as a reference laboratory, where troublesome blood samples would be sent for us to help analyze, allowing me to develop various protocols and troubleshoot problematic samples from hospitals across Taiwan.

During this time, I attended a workshop on red blood cell genotyping where I met a helpful professor from Lund, Jill Storry. The workshop sparked several questions, leading to an exchange of contact information to continue the conversation via email. Afterward, I realized that this was someone from the lab whose work I’d been reading and following for the past few years when trying to develop things at the blood center. At the time I felt that I wanted to continue learning and improving, so I asked whether there was a possibility to join their lab as a PhD student. Later, when they had an open position, they asked whether I might be interested in applying. So, that’s how I ended up in Lund and now I’ve been at the University and Lund Stem Cell Center for the past four and half years,” answers Gloria Wu.

Can you tell us about the cover of your thesis?

“Oh yes, my thesis cover, I made it out of desperation. Every time I could not write anymore during the thesis writing process, I would try to make a cover. This went on for several weeks and I just could not come up with something that I was happy with. I do not have much talent for drawing, so when someone suggested using AI-generated art, I gave it a try. And I am very happy with the outcome, it is a very “fun” cover, just as the journey of the PhD study.

I feel this cover represents what I wanted to convey throughout my thesis which is entitled: “Elucidating the Blood Group Regulome,” or how the blood group genes are regulated. That's what we set out to understand at the start of my doctoral studies, but then during the four and half years, all I began to do was only scrape the surface. My thesis only reveals a little bit of light on the whole of blood group regulation. So, on the cover of my thesis is the DNA helix with some shapes surrounding it, set against the dark blue sky. What my work has done, is to shine a simple light with these little stars into the darkness of the dark blue sky – which represents the unknown.

I chose this image as well because it captures my love for star gazing and astronomy. I always tell people that my hobby is astronomy, and my profession is blood groups, which are the two fundamental things that people like to use to characterize people. They characterize people with the stars using astrology and the zodiac signs, and then with blood types usually grouping into four different types – A, B, AB, O. So, I'm the right person if you want to discuss those things,” jokes Gloria Wu.

What have you found the most enjoyable during your Ph.D. studies? 

“I've found great joy in the richness of lab life. Our lab environment is top-notch with numerous collaborations, ample funding, and intriguing cases from the clinic. Working under my supervisor, who is also the Medical Director of the Nordic Reference Laboratory for Genomic Blood Group Analysis, located in Lund, allows us to investigate complex cases from the clinic which engenders interesting ideas itself, but is also good to see how people problem-solve in different ways. The exposure to diverse techniques and problem-solving approaches is truly enlightening and enriching, and I tried to soak that in.

I’ve learned so many new things and was able to implement some of my ideas into the project. Learning in this interactive manner, where I took in information and contributed my insights, was quite enjoyable. I thought that balance was very nice. 

Even amid the challenges of the pandemic, which began just six months after I arrived in Lund, I was lucky to have a very supportive and very friendly group of colleagues. We organized social activities to stay connected, maintain a sense of community, and uplift our spirits during the pandemic. This period might have been the highlight of my PhD studies, surprisingly.

Throughout the pandemic, everything came to a halt, yet everything turned out well. I was fortunate to have supportive colleagues who guided me through, looking back it feels like we successfully navigated these challenges together,” highlights Gloria Wu.

What has been the most challenging aspect?

“However, challenges do arise, both in studies and personal life. Being from Taiwan, a subtropical island in the southern Pacific, the dark, cold winters here in Sweden have presented some difficulties. To cope, I have adopted the Swedish strategy to overcome winter - escaping to nearby locations that are warm during the winter months.

Also, considering English is not my first language, sometimes, conveying my ideas clearly can be a bit challenging. Then, of course, cultural differences add another layer of complexity. In conversations here, people tend to be reserved, which in the beginning left me uncertain at times. However, I have learned the way over time – being direct and making sure I confirm my understanding,” notes Gloria Wu.

What are your plans following your Ph.D. defense?

“I would love to stay in this field. I loved my work as an MT back home, and plan to pursue a similar position here in Sweden, possibly a role that is clinic-based. To do that means, that learning Swedish will be a top priority following my defense. I also plan to relocate to Uppsala, further north in Sweden, where my husband is located,” shares Gloria Wu.

Any other tips or advice for future Ph.D. students? 

“I would say be open-minded. To not be afraid to make mistakes, because that is the best way to learn. Take all the opportunities for learning that the University offers in general and especially at the Lund Stem Cell Center - which is a lot. I not only mean taking advantage of accessing the core facilities but also the mentorship programs and career programs for PhD students, it's amazing.

I was part of the Professional Development Program (PDP) at the Center and the University’s MentiLife program, and I can strongly recommend future students to utilize these amazing resources and take part in these two programs. I think they help you to first grow as a person, and then second to deal with all the difficulties that arise during your studies. You’ll be much better off if you have all those supports in place,” highlights Gloria Wu.