Searching for the Rare Variants in a Genetic Haystack

In August 2018, immunologist Carola Vinuesa was working in her office at the Australian National University (ANU) when she received an unexpected call from David Wallace, a former student. He wanted to discuss Kathleen Folbigg, an Australian woman who had been convicted 15 years earlier for manslaughter of her first child and murder for her other three. 

Her interest piqued, Vinuesa listened as Wallace described how, over a ten-year period, each of Folbigg’s children, aged 19 days to 18 months, died in their sleep. The death certificates of the first three children—Caleb, Patrick, and Sarah—stated that their deaths resulted from natural causes. However, it was following the death of their fourth child, Laura, that the Folbigg’s case reached the local constabulary. Despite evidence of myocarditis on autopsy, potentially caused by a concurrent respiratory infection, the forensic pathologist who performed the autopsy recorded the cause of death as undetermined when alerted to the three previous deaths in the family. This decision opened the door to a potential case of infanticide. 

The Folbigg’s situation was not only devastating, it was highly unusual—one death in the family was tragic, but four? In 1999, Kathleen’s husband Craig incriminated her after finding distraught diary entries in which she exhibited guilt and self-blame, which is not uncommon among grieving parents. Four years later, she was sentenced to 40 years in prison. 

“[Wallace] said [that] he tried to call a few other people to see if they would be interested in what he had to say and he hadn’t been able to elicit a lot of interest,” said Vinuesa. She listened as he outlined concerns raised by Folbigg’s legal team regarding the evidence provided to the jury, which they felt was circumstantial and misleading rather than direct evidence of foul play. Wallace was not involved in the case, but he had studied it during law school. He reached out to Vinuesa to see whether an underlying genetic condition could explain the sudden deaths. 

In addition to her established research career and expertise in genome sequencing, Vinuesa is also a medical doctor. When she read about the myocarditis, epilepsy, and respiratory infections reported around the time of the children’s deaths she became suspicious—not because of the improbability of four deaths in the same family, but because it seemed entirely plausible that the deaths had resulted from genetic mutations, not murder.  

Intrigued by the case, Vinuesa began working with Folbigg’s legal team and other scientists to sequence the family’s genomes in search of variants that could possibly explain these tragic deaths. Her discoveries would change the course of Folbigg’s life and ignite a broader conversation about using genetics in the courtroom.

Vinuesa’s scientific career has taken her around the world and back. She’s delved into the depths of the genome and launched research centers dedicated to unraveling the mysteries of human genetic variation in disease. Since 2021, she has led a research group at the Francis Crick Institute, not far from where her research career began 30 years ago.

Journey to the Center of the Lymph Nodes

A desire to improve the lives of others has always been a guiding light for Vinuesa, who attended medical school in Spain, where she was born and raised. During her studies, her work with different nongovernmental organizations took her to a leprosy clinic in Kolkata and clinics in rural Ghana. 

“It was quite frustrating because most of the infections we were dealing with, we did not know why the children were dying, or why we couldn’t treat them any better,” said Vinuesa. She viewed the problem as the result of a bigger gap in scientific understanding of why the immune system struggles to fight infections like pneumonia or meningitis that are caused by polysaccharide-coated bacteria, or why vaccines work against certain pathogens but not others. 

In the early 1990s, Vinuesa completed medical school and moved to the UK for a residency program, but she soon found that she was dissatisfied and wanted to change gears. She enjoyed formulating hypotheses and dissecting mechanisms, and the scientific questions that started popping up during her medical training still lingered in her mind. She followed her intuition and enrolled in an immunology PhD program at the University of Birmingham, where she worked with immunologist Ian MacLennan on immune responses to encapsulated bacteria.

“I just simply loved it,” said Vinuesa, who quickly found that the research life was a perfect match for her. “I found this is really what I want to do. It could eventually save more lives than just working as an individual doctor in a particular place.”  

She dove headfirst into the study of immunity, where she would discover a career-long interest in the T cells and B cells that orchestrate antibody production. She homed in on a key production site: germinal centers. 

Following an infection or immunization, many types of immune cells converge in the lymph nodes to prepare for battle. Antigen-presenting cells deliver intel on the “pathogen non grata” to teams of T cells and B cells. In turn, the helper T cells converse with the B cells to support the B cells’ responses to the foreign antigen.¹ Some of these B cell cadets are then shipped to specialized pop-up training facilities, or germinal centers, which are located at the edges of the lymph nodes.² Here, they proliferate and undergo somatic hypermutation: the introduction of point mutations that increase or decrease a B cell’s specificity for a foreign antigen. Poorly performing B cells get the ax while high performing cadets are promoted to the ranks of long-lived antibody-producing plasma cells, or memory B cells, and are released into the bloodstream where they play an important role in the adaptive immune response.

In contrast, encapsulated bacteria directly activate B cells and stimulate antibody production in a T cell-independent fashion. “The dogma was that you do not get germinal centers against these types of bacteria,” said Vinuesa. She found that these activated B cells proliferated outside of germinal centers in extrafollicular regions, but she also showed that, in certain conditions, germinal centers could form without the help of T cells.^3-5 Furthermore, although T cells are not required to activate B cells to form germinal centers in response to polysaccharide antigens, they are essential for sustaining these germinal centers. These experiments further piqued her interest in the helper T cells and their behaviors both inside and outside of germinal centers. 

“[These findings] have always been recognized as important immunology research, but I think that more recently, because of a confluence of work from different groups, it’s become obvious that it is very important in autoimmunity, and in particular human autoimmunity,” said Ignacio Sanz, an immunologist at Emory University.

Following her PhD, Vinuesa received a Wellcome Trust International Research Fellowship which took her to the other side of the world—Canberra, Australia—where she would enter the forefront of genome sequencing in human disease. 

A Medieval Saint and the Naming of a Novel Gene

When Vinuesa finished her graduate studies, scientists were still working on the draft of the human genome, but she was eager to begin exploring the intersection of immunology and genetics. An ambitious program run by immunogeneticist Christopher Goodnow at ANU caught her attention. Goodnow was using chemical mutagenesis to introduce millions of random mutations into mice in order to pinpoint the genes that regulate immunity.⁶ Vinuesa joined Goodnow’s team and got to work running large-scale screens to identify which mutations caused mice to develop autoimmune responses. Several genes emerged as regulators of autoimmunity, including a novel gene with a previously unknown function.  

“I was very excited,” said Vinuesa. “It was the discovery of a new gene, [and] I could name it.” 

For this, she received a bit of divine inspiration. Where Vinuesa grew up in Spain, Saint Roch (San Roque in Spanish) is a big deal. Saint Roch garnered celebrity in the mid-fourteenth century for curing victims of the bubonic plague after surviving the deadly disease himself. When he was ill, he developed large buboes, or inflamed lymph nodes, a phenotype shared by the mice that carried a mutation in the gene that she named roquin.⁷ 

Vinuesa found that the roquin mutation caused a severe lupus-like autoimmune disease in the animals, which she named sanroque mice. Somehow, germinal centers were forming in the absence of a foreign antigen. She discovered that the mutation set off a molecular chain of events that resulted in an accumulation of helper T cells inside the germinal centers and the production of autoantibodies. 

In 2006, Vinuesa became a group leader at ANU, where she continued to dissect the molecular mechanisms by which the roquin gene regulates the helper T cells that camp out inside germinal centers.⁸ Subsequent research by Vinuesa’s team, in combination with work from two other groups, was pivotal for establishing these germinal center T cells, or follicular helper T cells, as a distinct subset of T cells that are required for systemic autoimmunity.^9-11 “They were the true helpers of B cells,” said Vinuesa.

Vinuesa had just started her own laboratory at ANU, but she was already showing the signs of a seasoned mentor. “Carola is a very motivating supervisor,” said Di Yu, Vinuesa’s first graduate student trainee who is now an immunologist at the University of Queensland. Yu noted that her positivity and enthusiasm was ever present and guided him through some of the tougher moments in science, such as responding to negative experimental results, and her ability to champion and empower her trainees is something that he tries to emulate with his own students. 

“She’s managed to be this unicorn scientist who does phenomenal research that genuinely has changed people’s lives, but she does it whilst being a phenomenal human being as well,” said Michelle Linterman, an immunologist at the Babraham Institute and one of Vinuesa’s former graduate students.  

Bespoke Mouse Models Informed by Human Genetics

The random insertion of mutations into the mouse genome led to the serendipitous discovery that the roquin gene plays an important role in immunity, but to better understand human disease, Vinuesa knew that she needed to go straight to the source. It was the late 2000s, and with the blueprint of the human genome in hand and access to new sequencing tools, Vinuesa set out to find disease-specific gene variants in humans that she could take back to the bench to give her mouse models a makeover. “As soon as we had the tools to do it in humans we moved very swiftly because, for me, it was very important to know that our research was relevant to humans,” said Vinuesa.

Although Vinuesa’s work broadly focuses on autoimmunity, she has made several strides in the genetics of systemic lupus erythematosus (SLE), a complex, multiorgan condition with no single cause or symptom profile. There is no cure for the disease and treatment is largely limited to broad immunosuppressants, starting with steroids, that can cause serious side effects and fail to treat the most severe symptoms.

Vinuesa said that she is proud of the project that she and her colleagues took on in these early days of whole-genome sequencing. “We were pioneering,” she added. Not only were they among the first to use genome sequencing in humans, they took on the challenge of finding rare variants in humans.¹² “At the time, it was not a very popular thing to do in autoimmunity,” said Vinuesa, who noted that the prevailing thinking at the time argued that autoimmunity was polygenic and that conditions would arise from a combination of multiple common variants with individually weak effects. 

Despite criticisms from others in the field, Vinuesa persisted with her efforts. “Our intention was not to solve the genetic architecture of lupus,” said Vinuesa. Instead, she hoped that by identifying rare variants that had functional consequences, then she could use this information to identify new therapeutic targets that are relevant for human autoimmune disease more broadly. “I always cite the example of the cholesterol receptor,” said Vinuesa. Biochemists Michael Brown and Joseph Goldstein discovered the low-density lipoprotein (LDL) receptor while unraveling the genetic culprits of familial hypercholesterolemia, which causes elevated blood cholesterol levels and an increased risk for heart attacks.¹³ Around one in 300,000 individuals carry the rare and life-threatening homozygous form of the LDL mutation that fueled the duo’s research.¹⁴ However, their findings helped uncover the importance of cholesterol metabolism in cardiovascular disease, and eventually led to the prescription of cholesterol-lowering treatments to patients at risk of developing cardiovascular disease regardless of whether they have the LDL receptor mutation. 

Vinuesa sees parallels with her own research exploring mutations that contribute to the pathogenesis of autoimmune disorders. “By finding some of these very penetrant, rare variants with strong effects, we might find fundamentally important pathways that would be relevant to the vast majority of patients,” said Vinuesa. 

In 2014 she became the founder and co-director of the Center for Personalized Immunology at ANU. One of the Center’s goals is to identify rare genetic variants in patients with autoimmune disorders and test them in the laboratory using bespoke mouse models that facilitate causal experiments. 

After identifying rare missense variants in the P2RY8 receptor—previously identified as a regulator of B cell migration in germinal centers—in a subset of patients with SLE, Vinuesa and her team generated mutant mice that overexpressed the receptor, which allowed them to uncover a broader role for the receptor in immunological tolerance.¹⁵ Vinuesa has used this inverted approach to further demonstrate the role that several other rare variants and gain-of-function mutations contribute to human lupus and complex immune deficiency.^16,17 

In 2023, Vinuesa received the Lupus Insight Prize for the discovery that gain-of-function mutations in the gene that encodes the toll-like receptor 7 (TLR7) cause human lupus.¹⁸ Her team identified a de novo missense variant in a seven-year-old child presenting with severe lupus and found that the mutation was sufficient to cause lupus-like symptoms when introduced to mice. Using these genetically modified mice, they showed that the mutation caused increased TLR7 signaling which allowed self-reactive B cells to survive.

Sanz’s team at Emory University previously found that a subset of autoreactive B cells was activated in patients with lupus and identified TLR7 as a major driver of these immune responses, but it was a chicken-or the-egg situation until Vinuesa and her team provided the first evidence that a mutation in the TLR7 gene directly targeted B cells to drive lupus pathogenesis. 

“It came together to really make a very convincing case…that, in fact, the B cells are central to lupus, and that many of the mechanisms are related to how TLR7 may actually dysregulate B cells and generate lupus,” said Sanz. “That’s a very important contribution.”

Harkening back to lessons taken from Brown and Goldstein, Vinuesa said, “Even though a minority of patients might have these variants, this pathway is central to lupus pathogenic disease in humans, and I think it’s the most important pathway in the vast majority of lupus.” Whether or not a patient has a TLR7 mutation, TLR7 inhibitors are available and pharmaceutical companies are currently exploring their potential in phase II clinical trials. “We are very hopeful to see the results of those trials at the end of this year or beginning of next year,” said Vinuesa, who is not involved in the trials herself. 

Even as Vinuesa made significant progress in understanding the genetic causes of lupus and other autoimmune diseases, her sense of duty would not let her turn away from someone she was uniquely positioned to help. So, when she received that out-of-the-blue phone call in 2018, she quickly joined forces with Folbigg’s team to determine whether there was exculpatory evidence in the exome. The five-year legal battle about to ensue would demand a resolute and unswerving leader like Vinuesa. 

Genome Sequencing Gets its Day in Court

Following her conversation with Wallace, Vinuesa spent a few days looking into the Folbigg case. After reading through the medical records of each child, she felt that the clinical symptoms—floppy larynx, a potential respiratory infection, myocarditis, epilepsy—suggested a natural cause of the deaths that could be explained by genetic mutations. Plus, cherry-picked diary entries meant to depict a distraught and malicious mother, along with the prosecution’s use of shaky statistics and ill-informed guidance from medical experts that sudden infant death syndrome did not recur in families, didn’t sit right with her.¹⁹ 

Despite the picture painted by the prosecution in the Folbigg case, Vinuesa knew it was possible. By chance, one month before Wallace contacted Vinuesa, she was referred a medical case about a family from Macedonia that had suffered the loss of four infant children. Vinuesa identified the genetic causes of their deaths. “These deaths occur—four deaths in a family is not unheard of,” said Vinuesa. 

When Vinuesa and the legal team set off in their search of genetic mutations that could similarly provide clues about the deaths of the Folbigg children, they ran into a roadblock: they didn’t think that they would be able to get a court order to access genetic material from the children. Vinuesa reasoned that if there was a genetic cause it could be inherited from either the mother or father. A simple saliva sample could begin to shed light on the case. “We decided to start from the mother in the very long shot that we could find something there,” said Vinuesa, “which eventually we did.”

Vinuesa and her team found that Kathleen carried a novel calmodulin variant (CALM2 G114R).²⁰ Calmodulinopathies are rare conditions that can cause life-threatening arrhythmias and they result from pathogenic variants in one of three CALM genes: CALM1, CALM2, or CALM3.²¹ When they gained access to genetic samples from the children, they found that both Sarah and Laura carried the CALM2 variant. Although the daughters and Kathleen had the mutation, the mother’s symptoms were mild—she reported an occasional fainting spell following exertion. However, scientists have found that a calmodulin variant can have differential clinical manifestations in different individuals, even within the same family.^21,22

In a report submitted as part of a judicial inquiry into the convictions, Vinuesa and her team concluded that this variant was the likely cause of their deaths; however, the commissioner did not find reasonable doubt. To prove that this variant was arrhythmogenic, Vinuesa teamed up with biochemists in Denmark who, together with collaborators in the US and Canada, demonstrated that this variant, like two other known CALM pathogenic variants, affected the function of calcium channels that are critical for regulating heart contractions. 

Although the two sons did not carry the CALM2 variant, Vinuesa and her team found genetic fingerprints that suggested that they carried potentially pathogenic rare missense variants in the Bassoon gene. Although they did not run follow-up analyses to determine whether the rare variants played a causal role in their deaths, research in mice suggests that variants in this gene can cause early onset lethal epilepsy.²³ “That wasn’t necessary because just by proving that the two daughters had an arrhythmogenic variant that would have caused their sudden death, that was enough,” said Vinuesa, who noted that there was already sufficient clinical evidence that said that the two boys had died from natural causes. 

“People think this is rare, but in genomics clinics, five percent of all families actually have two Mendelian conditions. These are the problems that we face,” said Vinuesa. “People think it might be rare, but rare diseases are not that rare.” 

In March 2021, Folbigg’s legal team submitted a petition, signed by more than 100 scientists, including Vinuesa, to the Governor of New South Wales to pardon Folbigg. They argued that the evidence used to convict Folbigg was circumstantial and that the CALM2 variant was the likely cause of death of Sarah and Laura Folbigg. Ultimately, they were successful: In June 2023, 20 years into her 40-year sentence, Folbigg was released from prison.

“It wasn’t easy,” said Vinuesa. It took five years, two legal inquiries, and contending with a lot of opposition to get to this point. But Vinuesa said that it was a cause worth fighting for. “It became important to me to try and show that science can solve important problems in society,” said Vinuesa. 

One pushback that Vinuesa and her team faced when analyzing the genetic data in the Folbigg case came from scientists who wanted to apply the same strict framework established by the American College of Medical Genetics and Genomics to help clinicians determine the likelihood that a gene variant is pathogenic.²⁴ “These are not clinical cases where the result of your diagnosis is going to inform whether a woman should have a mastectomy or not,” said Vinuesa. Doctors want to have a high level of certainty—90 percent or higher—about whether a gene mutation is going to cause cancer before taking drastic interventions. “But in these court cases where all you’re trying to find is reasonable doubt or whether there is a reasonable possibility that a mutation can have caused the death of children, and is not going to trigger clinical action, we can’t use the same rigid framework that we are using in clinical diagnosis.” Variants that fall below this threshold are being ignored or disregarded, said Vinuesa. “It is a problem, a huge problem, and I’ve become very interested in this area, not just in terms of justice for poorly convicted mothers, but in terms of the need to educate pediatricians when they make these diagnoses.” 

Saint Roch, as well as lending his name to the gene discovered by Vinuesa, is a patron saint of the falsely accused. “That she picked this [case] up just speaks to her sense of justice and integrity,” said Linterman. “She has an extremely high level of resilience in those cases, because she very much knows what she believes to be right, and she’s willing to fight for it.”

Vinuesa’s sedulousness goes well beyond the Folbigg case. Her scientific journey around the world over the last three decades is distinguished by a dogged determination to identify and understand rare events and an admirable compassion towards those suffering from diseases of unknown causes.

“I’ve always considered Carola as a person with a really big heart,” said Yu. “She is a hero in my world—to sacrifice her time [and] demonstrate the responsibility as a scientist of what we need to do if we can.”

SusanneB Vinuesa was nominated for this profile through The Scientist’s Peer Profile Program submissions.