How CIRCA works: a summary

Candidate families for sequencing are presented by their consulting immunologists and screened on the basis of strict criteria, including extensive phenotyping and sequencing of a panel of known genes.

1. Patients with extraordinary clinical presentations and strong family histories are recruited to take part in CIRCA.
2. Targeted sequencing or diagnostic whole genome sequencing is then performed through the Immunopathology lab at the Children's Hospital at Westmead. 
3. The function of a patient’s immune cells is assessed by affiliated research groups with the capacity to develop mouse models of disease using the CRISPR/Cas9 platform.
4. Potential disease causing genes are identified; where known disease-causing mutations are not found, the sequence will be interrogated for novel mutations.

Next steps: defining new molecular pathologies

Following genetic analysis, consulting clinicians, geneticists and research scientists collaborate to identify candidate gene variations and perform functional studies. These include in vitro human cell assays and establishment of mouse models with orthologous mutations, to test the functional consequences of the genetic variations observed.

This experimental work has the power to confirm which genetic variations are causative of disease, and through what mechanism.

Background information

Every individual’s genome contains large numbers of genetic variations, so understanding which of a patients’ gene mutations actually causes disease requires in-depth analysis in experimental systems.

An individual’s genetics might also influence their response to immunotherapies in the context of cancer treatment or organ transplantation.

Primary immunodeficiencies (PIDs) provide clues to immune function

A key focus of CIRCA is “primary immunodeficiencies” (PIDs), which are a collection of rare, chronic disorders, in which important cells of the immune system either fail to develop or the immune system functions improperly. Individuals with a PID are susceptible to frequent and potentially fatal infections with a range of pathogens. PIDs are often also associated with autoinflammatory disease, as well as the development of cancer.

PIDs are commonly diagnosed in early childhood and arise because of genetic variation. PID-associated genetic variants have already been identified in nearly 300 different genes; however, the exact cause of disease in many PID cases is not yet known. Because PIDs arise from a range of different gene variants, the symptoms of the diseases may differ.

Because the genes involved in PIDs are generally critical regulators of the immune response, the study of PID-associated genes has led to a greatly increased understanding of immune function in general. This knowledge is also transferrable to other more common conditions.

De novo mutations and the pathogenesis of childhood-onset autoimmune disease

An autoimmune disease occurs when a person's immune system mistakenly attacks their own body. Genetics are likely to be a major contributor when children develop autoimmune diseases the first ten years of life. In a study funded by the National Health and Medical Research Council, we are testing the hypothesis that many of these cases are caused by de novo mutations in DNA (genetic “mistakes” present in a child, but not their ancestors) that disrupt the mechanisms normally preventing the immune system from attacking the body.

The project will bring together pediatricians and clinical specialists in childhood-onset autoimmune disease with experts in the analysis of genes and cellular mechanisms for self-tolerance and autoimmunity. Two new technologies make testing these hypotheses possible for the first time: whole genome sequencing on the Illumina X-Ten platform, and testing of candidate mutations by introducing them into the mice using CRISPR-Cas9.

The genome sequence of patients and both their parents will be examined, in collaboration with endocrinologists and pediatricians at Westmead and Sydney Children’s Hospitals. Genetic variations that are observed will be validated experimentally by introducing those mutations into the mouse models. These bespoke mouse strains will be used to define exactly how the mutations disrupt immune function, and to test pathway-specific therapeutics. The immune cells present in the patients blood will be studied in order to develop laboratory tests for genetic lesions in specific pathways resulting in autoimmune disease.

The power of genetic diagnosis for rare immune disorders

People with rare, clinically extreme conditions of immune dysregulation may find it difficult to obtain a diagnosis through traditional clinical testing methods. For these patients, undergoing genetic testing or genome sequencing may make it possible to obtain a genetic diagnosis (an identification of the genetic variant that underpins their disease).

A genetic diagnosis makes it possible to tailor support or treatment specifically to the individual, and to anticipate future concerns. It can also bring to an end the ‘diagnostic odyssey’ of invasive, inconclusive testing.

For the broader community, the information gained from understanding the genetics of rare conditions can also reveal much about the workings of the immune system. This type of information has been immediately transferrable to more common immunological diseases. For example, the discoveries of disease-causing  genes in cases of severe immune dysfunction (TNF alpha receptor, IL-1 receptor antagonist and JAK3) have given rise to new therapeutic targets for rheumatoid arthritis.