What Two Sisters With a Rare Heart Condition Taught Doctors About Our Genes

Early in February of 2008, just days after she was born, Tatiana Legkiy lay in a cardiac intensive care unit, her tiny body hooked up to a respirator. After crying for two hours, she was now briefly quiet, the tube in her throat helping her breathe but also preventing her from making any sound.

Tatiana’s heart was failing. A cardiologist, tipped off by a pediatrician who heard something strange in a routine checkup, had examined her earlier that day and grown worried. He sent Tatiana to a nearby hospital in Modesto, California, where she remained for only an hour before being whisked eighty miles west by ambulance to the UCSF Benioff Children’s Hospital.

Tatiana’s parents arrived at the hospital shortly afterwards. At the time, Lana and Andrey Legkiy lived in Manteca, a city in California’s Central Valley. Andrey worked at an animal supply company; Lana stayed home and took care of their four-year-old daughter, Anna. Weekends found the the family outside together, camping or fishing in the Delta where the San Joaquin and Sacramento rivers flow into the ocean.

Even before Tatiana’s birth, the Legkiys knew medical hardship well. A year earlier Lana had suffered a miscarriage, losing an unborn child, a boy, in her third trimester. At the time, physicians had ascribed his cause of death to pulmonary hypoplasia, or incomplete development of the lungs.

Given that medical history, when Tatiana arrived at Benioff in critical condition, the doctors requested slides from the unborn child’s autopsy. This time, taking a closer look at small samples of heart tissue, they noted the true cause of death — an extremely rare heart condition known as left ventricular noncompaction (LVNC), in which the heart muscle remains immature and cannot pump blood normally.

Visually, physicians identify LVNC by the fingerlike protrusions of muscle extending from the wall of the heart into the left ventricle, which supplies the body with oxygen-rich blood. An echocardiogram of Tatiana’s heart showed these same characteristics.

Gently, the physicians told Tatiana’s parents that there would be no surgery, but only because LVNC has no cure.

A Hunch

Immediately, Deepak Srivastava, then-attending physician at Benioff, suspected a genetic connection. He was a geneticist, after all, and Tatiana’s unborn sibling had shared the same disease.

Though he could not have foreseen it as Tatiana struggled for her life that day, proving this intuition would require over a decade of work. It would also require technology which was only just becoming usable, and the dedication of researchers he had not yet met.

In the coming years, Srivastava would move forward with his own career, juggling the roles of biology professor, pediatric cardiologist, and ultimately president of Gladstone Institutes, a nonprofit biomedical research institution in San Francisco.

But the story of the Legkiys would stay with him. For a decade, he wouldn’t be able to shake the desire to pinpoint a precise genetic cause of LVNC, a necessary first step towards finding a cure for the disease. With that knowledge, physicians could rapidly screen potential drugs using an accurate, personalized model for patients like Tatiana.

Adding to the urgency of the Legkiys’ situation: Tatiana was not their only child. Even as Tatiana received her diagnosis in 2008, her happy four-year-old sister, Anna, ran around the hospital waiting room. Blissfully oblivious, Anna charmed the physicians, who began to wonder: did all three Legkiy children share the same disease?

As Tatiana stabilized, thanks to medication helping her heart pump and a respirator helping her breathe, Srivastava’s team set about finding an answer. Two days after diagnosing Tatiana, the scientists took an echocardiogram, a kind of ultrasound, of her father’s heart. They observed that Andrey had a less severe, asymptomatic case of Tatiana’s condition.

So by the time they tested Anna’s heart the following day, Lana Legkiy already knew more than a mother should about what LVNC looked like in an ultrasound. (The team would later examine Lana’s own heart, and find it normal.)

When she saw Anna’s heart up on the screen, she knew without being told that her daughters shared the same disease.

Today, Lana can’t remember precisely what she felt in that moment— so much happened at once — but she does recall a dream she had while in the hospital, after Tatiana’s diagnosis. Lana doesn’t consider herself a true believer of supernatural occurrences, but she thinks dreams are important. “I saw both of them… as grown women,” she said. “I think that settled me down a little bit, like, ‘Okay, we’re going to be okay.’”

Srivastava, however, remembers the Legkiys’ distress acutely. “It was actually very sad,” he said. “This is a family who had lost one child, their other child was in a life-threatening situation, and then they find out their other girl has the same thing… it was actually pretty devastating.”

The Long Search for Proof

As the physicians told the Legkiys, there was and is still no cure or surgical procedure that can remedy LVNC. In cases such as Anna’s, the heart compensates for its left ventricle and swells, allowing it to pump more blood through the body. Anna, like others with enlarged hearts, did not display typical symptoms of heart dysfunction such as fatigue or rapid breathing.

Srivastava, however, told the Legkiys that both girls’ hearts would need to be monitored with yearly ultrasounds. Tatiana was sent home with more of the medication that helped her heart pump; she would take the medicine for a year before her heart muscles improved and the medication was no longer necessary. The family resumed what was, for the most part, a normal life.

As Anna and Tatiana grew into young women, now 15 and 11, Srivastava and his coworkers set about pinpointing the precise causes of the family’s congenital heart disease. The team sequenced the family’s DNA, combing through their genes in search of the variants all three children shared, screening against frequency of occurrence in the general population and for association with the heart.

The scientists quickly found three mutations in three different genes shared by the three siblings that they suspected to be the cause of the disease, two inherited from Andrey (MKL2 and MYH7) and one from Lana (NKX2-5). If not for the advent of a few key technologies, the investigation would have stopped here, the Legkiys left to wonder what was hidden in their genes. Srivastava’s search for proof, however, was just beginning.

CRISPR’s Perfect Timing

By 2014, the Legkiys were living in Seattle; Tatiana and Anna were six and 10, both medication-free. Tatiana’s hospital experience seemed like a distant memory.

In San Francisco, meanwhile, Casey Gifford, a recent PhD from Harvard, had joined Srivastava’s research team. New to both human genomics and heart disease, she and Srivastava spent a four-hour car ride to a conference in Lake Tahoe discussing what they thought would be the next big questions in medicine. Srivastava brought up the family he couldn’t forget — and realized, as he did so, that the tools they would need to prove which genetic mutations caused LVNC in the Legkiys might finally exist.

CRISPR/Cas9, with which scientists can selectively remove and replace portions of the genome, had just been used in mammalian cells for the first time the year before. Its advent, Srivastava knew, meant that creating a mouse model with a desired genetic condition, which used to take a year, could now be done in three weeks.

Armed with CRISPR/Cas9, Srivastava and Gifford decided to engineer mice with the same three mutations that Srivastava suspected had caused the Legkiy siblings’ heart condition. They hypothesized that the mutations would result in phenotypes, or observable physical expressions of genes, that resembled the phenotype of the human disease. As predicted, the mice with all three mutations showed the same finger-like protrusions in their hearts, demonstrating that, at least in mice, the mutations were enough to cause LVNC.

A Patient’s Beating Heart (Cells)

The mouse heart tissue that resembled the Legkiy childrens’ was an exciting breakthrough, but Srivastava and Gifford knew that to persuade the scientific community of the genes’ importance, they’d need to design an equally convincing experiment in human cells. As Srivastava realized on that car ride, their timing could not have been better: in 2012, Gladstone researchers had won the Nobel Prize for induced pluripotent stem cells, cells that are genetically taken back in time to behave like embryonic stem cells, which scientists can then turn into any cell type in the body.

The scientists took skin grafts from the family and reprogrammed the cells to grow petri dishes full of heart cells genetically identical to Lana, Andrey, Anna and Tatiana.

In the lab, these cells pulsed (literally; heart cells pulse with a heartbeat all their own) in confirmation of the team’s diagnosis. Even these cardiomyocytes, the one kind of heart cell the researchers had decided to test, displayed hallmarks of LVNC.

Each family member had their own petri dish of cells. Lana’s cells spread across her petri dish the way normal cells do, as if they were stars in the sky. Anna’s, by contrast, clumped together and did not stick well to the dish. Together with RNA sequencing data, the results showed that cell adhesion, long suspected to play a role in LVNC, had been affected.

In conjunction with the CRISPR/Cas9 experiments, the cell study showed that the three mutations Srivastava’s team had identified had been enough to cause Anna and Tatiana to display symptoms of LVNC. This result, published in Science this May, gave proof to a phenomenon that had long been suspected by the medical community: that multiple genetic mutations could work together to cause a disease.

Lea Starita, a research assistant professor in the Department of Genome Sciences at the University of Washington, calls this proof “extremely important” for the field. She points out that the results hinged on the advent of cutting-edge technology and a single cardiologist paying the case a lot of attention.

“For years, I think we’ve known… that not all pathogenicities were going to be caused by single genes,” she said. “I don’t think there’s ever been a study that so well defined the interaction between three human variants on a phenotype like this.”

Looking Forward

Tatiana and Anna now live happy, normal lives: Anna is introverted and creative, a walking encyclopedia, her mother says; Tatiana is more outgoing. By now, their hearts seem to have developed normally, at last catching up to their bodies. Neither takes heart medication, though Srivastava says their hearts will need to be monitored for the rest of their lives, especially in moments of stress and when they get older. “It’s my hope that before they get to that point, we’ll have a better way to treat them,” he said.

With precise knowledge of which genetic mutations caused Anna and Tatiana’s rare heart condition, scientists now have taken the first steps toward identifying a cure. Gifford says that their combination of CRISPR/Cas9 models and use of stem cells illustrates the true power of genome editing. “We can [now] try to create much more faithful models of disease,” she said — models which could accelerate drug screening for a wide range of diseases, including other heart conditions and breast cancer.

The private sector has also been watching the Legkiys’ case, and is enthusiastic about what it could mean for how other genetic cases are studied. Tim Behrens, senior VP of a start-up company called Maze Therapeutics (not associated with the study), commends the work as “a terrific story of how genetics and functional genomics really [help] us understand the underlying biology driving disease.”

Behrens does point out that diseases influenced by many mutations would be harder to tackle. Informed by large-scale screening of genes for mutations, Maze is on its way to drug development with the hope that patients like Anna and Tatiana can receive treatment someday.

Srivastava, meanwhile, is still pushing the limits of what technology can do for patients. Advances in single cell assays from the past year are allowing his team to use CRISPR/Cas9 to test a hundred suspected mutations in patients’ cells at once. He says that they’re at work on the genomes of thousands of children with heart disease, all thanks to their experience with the Legkiys. “These cases are the kinds that don’t come along all that often,” he said, “but they’re the ones that can help us treat other diseases better.”