Novel Tools for Primary Immunodeficiency Diagnosis

Making a Case for Deep Profiling

Elena W.Y. Hsieh; Joseph D. Hernandez


Curr Opin Allergy Clin Immunol. 2016;16(6):549-556. 

In This Article

Abstract and Introduction


Purpose of review This review gives an overview of the systems-immunology single-cell proteomic and transcriptomic approaches that can be applied to study primary immunodeficiency. It also introduces recent advances in multiparameter tissue imaging, which allows extensive immune phenotyping in disease-affected tissue.

Recent findings Mass cytometry is a variation of flow cytometry that uses rare earth metal isotopes instead of fluorophores as tags bound to antibodies, allowing simultaneous measurement of over 40 parameters per single-cell. Mass cytomety enables comprehensive single-cell immunophenotyping and functional assessments, capturing the complexity of the immune system, and the molecularly heterogeneous consequences of primary immunodeficiency defects. Protein epitopes and transcripts can be simultaneously detected allowing immunophenotype and gene expression evaluation in mixed cell populations. Multiplexed epitope imaging has the potential to provide extensive phenotypic characterization at the subcellular level, in the context of 3D tissue microenvironment.

Summary Mass cytometry and multiplexed epitope imaging can complement genetic methods in diagnosis and study of the pathogenesis of primary immunodeficiencies. The ability to understand the effect of a specific defect across multiple immune cell types and pathways, and in affected tissues, may provide new insight into tissue-specific disease pathogenesis and evaluate effects of therapeutic interventions.


Primary immunodeficiencies (PIDs) constitute more than 300 genetic defects that alter the composition and/or function of the immune system. PIDs are considered rare diseases, but the true prevalence of each individual disease or all PIDs in aggregate is unknown. PIDs are molecularly and clinically heterogeneous,[1] representing an immune diagnostic and clinical management challenge. The variability in PID genotype–phenotype correlations complicates the ability to pursue candidate gene approaches. For example, patients with a similar clinical phenotype of autoimmune lymphoproliferative syndrome (ALPS) can have mutations in FAS, CTLA4, or PIK3CD genes.[2–4] Conversely, patients with the same homozygous mutations in RAG2 can present with different phenotypes, such as Omenn or hyper IgM syndromes.[5] Unbiased genetic approaches such as whole exome/genome sequencing (WES/WGS) have proven fruitful when educated guesses on candidate genes do not yield a unifying diagnosis. With these genomic advances, at least 10–15 new genetic defects associated with PID are identified yearly.[1] However, several factors confound the ability to diagnose PIDs, even with the use of WES/WGS. Deleterious mutations in noncoding regions and large structural variations such as deletions, inversions, and translocations, are often missed in WES/WGS because of inherent limitations of next generation DNA sequencing. Therefore, a 'negative' WES/WGS result, either with no identified variants or variants that are not consistent with the patient's phenotype, does not refute a diagnosis of PID.

Both WES and WGS generate substantial amounts of sequencing data, with the identification of multiple variants. Each exome has approximately 20 000 heterozygous and homozygous variants.[6] To narrow down the number of variants, several filters can be applied, based on suspected inheritance pattern and sequencing data from other affected family members. Additionally, bioinformatics programs such as Polyphen-2 or SIFT, can be used to predict the impact of a mutation on protein function.[7] After these analyses are applied, a list of variants is generated. Validation of these variants as disease-causing mutations still requires functional assays to explain patient-specific cellular and tissue pathophysiology.[8] Several methods have been used to evaluate functional consequences of newly discovered genetic variants, such as cytotoxic assays, knock-out/in mouse models, RNA interference, flow cytometry, and/or immunohistochemistry.[8] These approaches focus on one or few immune cell types and/or functional pathways.[8] In PIDs, however, perturbed immune processes may be pervasive across multiple immune cell types and signaling pathways. For example, in common variable immunodeficiency (CVID), defects in B-cell maturation, T-lymphocyte proliferation, and dendritic cell differentiation have been described.[9–11] Therefore, we need to consider the analysis of many immune parameters in many cells with single-cell resolution, essentially, deep proteomic and/or transcriptomic profiling. Deep profiling in PID can help to further pursue diagnosis in the setting of a 'negative' WES/WGS result or to validate variants identified by WES/WGS as pathogenic mutations.

This review will give an overview of systems-immunology deep profiling approaches to study PID. These tools can potentially help surmount challenges in: first, diagnosis, by uncovering novel immune defects; and second, clinical management, by identifying additional dysregulated immune cell types and pathways in currently known genetic defects, thereby directing therapeutic intervention.