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A life-changing diagnosis through genome sequencing.

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Alan's story

When 3-year-old Alan developed pink spots on his skin, his mum thought it was nothing more than his strawberry allergy acting up. But a trip to emergency revealed dangerously low levels of blood cell counts that led to years spent in hospital. His medical saga is not yet over, but the improvement in his condition has been immense.

Alan’s remarkable story is one of close collaboration between his clinicians at Sydney Children’s Hospital, Randwick and researchers at Garvan. It’s also a story of world-leading technology that is capable of rapid, high-quality genome sequencing and analysis, and of a dedicated search for answers by his parents and his clinical team.

He wasn't allowed to ride a bike, run, jump, or do normal things that children do. Any bleeding could be dangerous for him, deadly dangerous,” says Tansel, Alan’s dad. 

“Without Garvan, we would have lost Alan. We are now lucky that his condition is stable, and that his treatment continues to work. We are now waiting for a cure .” – Berna, Alan’s mum

Alan fought a battle for survival since age three. The first signs of trouble came while the family was based in Turkey. When Alan's parents questioned doctors about small red dots on his skin, tests revealed dangerously low platelet levels. “In that situation,” says Tansel, “if you have any bleeding, it's virtually impossible to stop.” Alan was rushed to hospital to treat this condition, thrombocytopenia.

Alan responded well enough to return home. But six months later his white blood cell count plummeted dramatically. Since these immune cells, called neutrophils, form the body's first line of defence against infections, this neutropenia left Alan very susceptible to infections.

Next, and worst, Alan's immune system began to destroy his red blood cells. This condition, haemolytic anaemia, is immediately life threatening. “We had to do whatever we can to stop it,” says Tansel, “and there's no way to do it unless you go to hospital, and he's under very serious care.”

Alan's immune system was aiming its arsenal of weaponry at normal parts of his body, instead of fighting infections. But why?

After the family returned to Australia in 2013, Alan came under care of doctors at Sydney Children's Hospital, Randwick; haematologist Dr Susan Russell, then paediatric immunologists Dr Brynn Wainstein and Dr Paul Gray.

His physicians searched intensively for the cause of Alan's perplexing disease. Suspecting it was genetic, they tested for known immune deficiency genes. But pinpointing the 'smoking gun' was extremely difficult, especially since many genes are unavailable for testing in Australia or globally.

Alan's autoimmune disease is extremely rare. But his experience mirrors millions of others' worldwide with monogenic disorders (also called Mendelian disorders). Caused by modification to a single gene found in all cells of the body, they lead to thousands of diseases, depending on the gene affected. Yet the underlying genetic cause is unknown in many of these diseases.

In spring 2015, Alan's autoimmune disease flared up again. By October, Alan had lost a lot of weight and lay extremely ill in the Intensive Care Unit of Sydney Children’s Hospital, Randwick.

Alan had severe diarrhoea and exceedingly low platelet counts. 

Dr Gray informed Dr Tony Roscioli, clinical geneticist at Garvan at the time, that the diagnostic quest was now extremely urgent.

Working through the sequencing data, Dr Roscioli scrutinised genes known to cause immunodeficiency. Within a couple of hours, he pinpointed the smoking gun: a gene called LRBA.

Alan's genome, the results showed, contained two exceedingly rare pathogenic (disease-causing) variants of the LRBA gene. Both Alan's copies interrupted its function.

Alan's healthy parents were silent carriers: each had one normal and one non-functional LRBA gene. Inherited singly in this way, the rare pathogenic variants could pass through generations without causing disease. Unfortunately Alan had inherited both non-functional genes, one from each parent. Doubled up, they caused his severe immunodeficiency.

Next, Dr Roscioli quickly confirmed the pattern of disease seen with LRBA deficiency matched Alan's immune problems. He also learned LRBA was on Alan's physicians' list of suspect genes. Remarkably, Tansel's own research had also led him to raise LRBA with the doctors. Yet LRBA deficiency is exceedingly rare.

The LRBA gene codes for the LRBA protein. This protein is found inside some of the body’s immune cells, particularly T regulatory cells.

The LRBA protein's job is to protect another molecule, CTLA-4, so it can build up on the outside of the cell. When LRBA is missing inside cells, they also lack CTLA-4 on the cell exterior.

These CTLA-4 molecules act as an important brake on the immune system. If this brake is missing, the immune system goes 'rogue', attacking normal body tissues, causing autoimmune disease.

Unfortunately, today very few genomic diagnoses for rare disease lead to direct therapies. These thousands of single-gene disorders, each with small numbers of patients, rarely enter drug companies' radar. However, genomics is helping bridge this therapy gap.

Even if there is no direct therapy at hand, genomic diagnoses have wide benefits.

Genomic results can also rule out further diagnostic testing, along with its discomfort, risk and cost. And even without a direct therapy, genetic diagnoses still help physicians better manage patients. They can refocus therapies and discontinue costly but unhelpful drugs, saving the health system millions.

Genomic discovery is also spearheading new therapies, yielding valuable insights into how genes function and, when they malfunction, the biological pathways causing disease. These insights suggest ways to re-purpose existing drugs, including new ways to treat rare disease.

This fast-paced world of genomic discovery delivered for Alan at a crucial time.

Dr Roscioli's findings marshalled the full clinical and research firepower behind the immunogenomics study. Professor Chris Goodnow at Garvan quickly investigated known findings on LRBA. Eight years earlier, he discovered, a child with a similar autoimmune disease was cared for at Cincinnati Children's Hospital in Ohio, USA.

Desperate for a treatment, the Cincinnati team had tried a new drug, Abatacept. Originally developed for organ transplant patients, Abatacept also helped with rheumatoid arthritis and psoriasis — both autoimmune conditions.

Abatacept, for reasons the Cincinnati team did not yet understand, rapidly corrected most of their patient's life-threatening symptoms. Over the next few years, they used Abatacept in two similar cases, again with strikingly positive results.

Then in 2012, UK researchers linked this spectrum of severe autoimmune problems to defects in a specific gene: LRBA.

The Cincinnati team subsequently learned that all three of their patients had this genetic LRBA deficiency. Joining forces with a team from the US National Institutes of Health they studied six more patients with LRBA deficiency, some also treated with Abatacept.

And they discovered why it works. Abatacept mimics the function of CTLA4, which is the crucial immune system brake lacking in LRBA-deficient patients. Abatacept compensates for this lack of CTLA4, restoring this vital brake. It prevents the immune system from directing its arsenal at normal body cells.

Published in July 2015, just three months before Alan's diagnosis, the US teams' discovery gave Professor Goodnow vital insight into biochemical workings of Alan's disorder — and a possible therapy.

With Alan gravely ill in ICU, his medical team urgently sought and received approval from the Sydney Children's Hospital, Randwick Review Board to use Abatacept. “All of this was done in record time,”says Tansel.

Alan began taking Abatacept in mid-October, funded through the Hospital. Cautiously optimistic, Alan's family and clinicians, together with a growing interdisciplinary team, watched, waited and hoped.

Alan recovered rapidly in the weeks after starting Abatacept. He was soon out of intensive care and into the children’s ward.

Alan, so recently gravely ill, was completely transformed.

Over the next half year, as Abatacept's effects built up, Alan's red blood cells and platelets rebounded into normal range. Haemolytic anaemia and thrombocytopenia, his most serious threats, are now under control. Alan is also less vulnerable to infection, able to cope without heading to emergency for antibiotics.

But Garvan researchers aimed to help turn Alan's partial fix into a complete one. Armed with Dr Roscioli's insights into Alan's LRBA gene, Professor Robert Brink at Garvan moved quickly to have Alan's very own mouse avatar produced.

Like avatars in movies and online games, avatar mice are stand-ins for real people. They allow medical researchers to study responses to drug therapies, to prove their safety and effectiveness. Using gene editing technology called CRISPR/Cas9, Professor Brink’s lab designs 'molecular scissors' to slice and modify genomes with remarkable ease and finesse.

Ready in February 2016, Alan's avatar mice mirror his genetic deficiency; their T regulatory cells lack the crucial CTLA-4 immune brake, tests show. Unlike Alan, however, these avatar mice are perfectly healthy, despite this missing immune brake. These mice are reared in a biobubble, isolated from infections — exposure to infections could be a critical factor. Could immune systems without LRBA stay on track if, like the mouse avatar, no infection pushes the immune system's accelerator pedal? The answers could reveal much for Alan and thousands of others with immune disorders.

The immunogenomics project began with discussions between Professor Goodnow and immunology colleagues who saw the potential of whole genome sequencing to illuminate the complex causes of immune disease and open the door for new treatments.

The Garvan Institute offered an excellent environment to bring their plans to life with its world-class technology, researchers and connections to hospitals. With the support of the Bill and Patricia Ritchie Foundation, Professor Goodnow began funding sequencing for patients with rare immune disorders in 2015.

They set up a pathway from the clinic to the lab, and back again. Just like in Alan’s case, the pathway begins when doctors identify patients with unusual immune disorders that they think could have a genetic basis. These patients are referred to have their genomes sequenced in the hope that this will provide a long sought after diagnosis and vital information for the management of their disease. Further lab work, like the use of avatar mice, provides additional insights.

Professor Goodnow believes that cases like Alan’s can have an impact beyond rare diseases.

The biological processes that make Alan’s condition life threatening are complex, yet the cause is easy to spot as he is missing one crucial gene. Many of the more common immune disorders, such as type 1 diabetes and rheumatoid arthritis, are much harder to explain.

By studying cases like Alan’s, researchers can better understand how the immune system works and why some treatments are effective. This knowledge can then be applied to the study and treatment of other disorders, potentially helping millions of people in Australia and around the world.

Alan is now an energetic teenager who trains at the gym with his friends and has started high school. He has been on abatacept for 10 years and is stable. Alan remains one of fewer than 500 people in the world identified with the rare LRBA variant.

Primary immunodeficiencies (PIDs) affect at least 6 million people worldwide, many of whom are children. Stem-cell transplants are a common treatment for severe immunodeficiencies but are invasive, can have complications and require lifelong immunosuppression. Garvan researchers are building on their world-leading expertise in CRISPR gene editing to advance therapy approaches for such diseases of the immune system. A team led by Professor Robert Brink is developing gene-editing therapies that can be administered to remove dysfunctional gene variants within stem cells, to effectively cure genetic immune diseases like Alan's. The next step is taking the research to animal testing.