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One gene, three mutations, two diseases

By Betty Zou  •  August 2, 2016


David Vetter was born in Texas in 1971 and spent the entire 12 years of his life in a sterile plastic bubble protected from the rest of the world. The media dubbed him “Bubble Boy” and his condition—severe combined immune deficiency—became known as “bubble boy disease.”

Our understanding of the disease and the genetic factors behind it has come a long way since Vetter’s diagnosis. Severe combined immune deficiency (SCID) disorders are caused by genetic defects that prevent the immune system from developing and functioning properly. New findings from a Sunnybrook-led team of researchers have shed light on the biological mechanisms underlying SCID and how different mutations in the same gene can lead to distinct forms of disease.

For the study, the researchers focused on a gene called RAG1. “The reason we examined RAG1 was because it has its own interesting biology,” says Dr. Juan Carlos Zúñiga-Pflücker, a senior scientist in Biological Sciences at Sunnybrook Research Institute, who oversaw the study. “It dictates DNA rearrangement—that’s the coolest thing there is in immunology.”

DNA rearrangement allows our bodies to generate an incredibly large and diverse repertoire of antibodies and immune cell receptors from a much smaller number of genes. By swapping gene segments, portions of DNA can be mixed and matched to generate up to one trillion different combinations, each encoding an antibody or immune cell receptor that recognizes a unique foreign substance. Immune cell receptors help the body decide if the particles it encounters are innocuous—like the salad you ate for lunch—or harmful—like the E. coli bacteria that might have contaminated the lettuce in your salad. RAG1 plays a crucial role in the process of DNA rearrangement by cutting up DNA fragments and rejoining them to create one-of-a-kind sequences.

To study the role of RAG1 in SCID, Zúñiga-Pflücker, who is also a professor and chair of the department of immunology at the University of Toronto, teamed up with Dr. Luigi Notarangelo, a pediatrician at Boston Children’s Hospital and professor at Harvard Medical School who specializes in SCID. They analyzed samples from two patients with SCID and one with Omenn syndrome, a subtype of SCID. Each patient had a different mutation in their RAG1 gene. The researchers used skin cells donated by the patients to make stem cells, which they then grew into T cells. T cells are white blood cells that play a critical role in immunity. A hallmark of SCID is a lack of T cells, which leads to immunodeficiency. By studying these patients’ cells, the researchers hoped to understand how their unique RAG1 mutations block T cell development and cause disease.

The team found that all three mutated forms of RAG1 were less able to rearrange DNA compared to a nonmutated or “wild-type” version of RAG1, essentially the form of the gene that occurs naturally. To facilitate DNA rearrangements, RAG1 cuts through both strands of DNA to generate a clean break before joining two ends together to form a new fragment. In the study, the researchers confirmed that the mutated RAG1 proteins were not able to cut through both strands of DNA as efficiently as their wild-type counterparts, thus lowering the rate of rearrangements. This impaired ability to mix and match DNA into new combinations led to blocks in T cell development and the immunodeficiency seen in these patients.

“One of the key results was actually finding the difference between SCID and Omenn syndrome [disease traits],” says Dr. Patrick Brauer, a postdoctoral fellow in Zúñiga-Pflücker’s lab and the study’s lead author. The mutated form of RAG1 from the patient with Omenn syndrome had lost its ability to cut double-stranded DNA but gained the ability to nick single strands of DNA. In contrast neither RAG1 mutations from the two patients with SCID exhibited this new function. “I was terribly surprised,” says Zúñiga-Pflücker about their results. “It was a very different set of blocks in development [in terms of] the stages in T cell differentiation that are affected in the absence of RAG1 activity versus in the presence of dysfunctional RAG1 activity.” These results provide important insights into how different mutations in the same gene can produce a wide range of immunological effects and lead to distinct disease characteristics in various patients.

Zúñiga-Pflücker notes that studying mutations in patients provides valuable information about human biology that cannot be gleaned from animal models alone. For example, until recent advances in gene editing, genetic manipulation in animal models was limited to knocking out an entire gene or dialling its expression up or down. Genetic mutations that occur in patients, however, are much more nuanced. They can slightly alter the protein’s shape to give it a new function, or cause it to be turned on or off at different times. Further, the same mutations do not always lead to the same outcomes in people and animal models. Studying these “naturally occurring” mutations in a human context deepens our knowledge of what happens in normal biology and disease. Zúñiga-Pflücker is continuing this work by examining the effects of different genetic mutations on T cell development in other patients with immunodeficiency syndromes.

This research was supported by the Canadian Institutes of Health Research, Krembil Foundation and National Institutes of Health. Zúñiga-Pflücker holds a Tier 1 Canada Research Chair in Developmental Immunology.

Dr. Juan Carlos Zúñiga-Pflücker