Variants of uncertain significance: a quandary in genetic testing
The contents of this article are informational only and are not intended to be a substitute for professional medical advice, diagnosis, or treatment recommendations. This editorial presents the views and experiences of the author and does not reflect the opinions or recommendations of the publisher of Rare Disease 360.
VUS findings can help us learn about rare immune disorders, but they don’t provide clear treatment directives. How should physicians respond to these results in the clinic?
By Jolan Eszter Walter, MD, PhD
This is part 2 of a 2-part series authored by Dr. Walter. Read part 1 here.
Rare primary immunodeficiencies have been notoriously difficult to diagnose, but that pattern is changing now that genetic testing has emerged as the quickest and most cost-effective way to distinguish between these conditions.
Accomplished through blood or urine testing, genetic sequencing can lead to more effective treatment and improved quality of life for patients with conditions such as activated PI3K delta syndrome (APDS) or autoimmune lymphoproliferative syndrome (ALPS), either of which can cause spleen and lymph node enlargement, cytopenias, and malignancy.1-6
Even as some physicians are embracing genetic testing, others are reluctant to introduce it because they don’t feel comfortable interpreting or acting on its results. A key concern involves mutations known as variants of uncertain significance (VUS), which can arise within disease-associated genes but are not linked to an illness by any scientific evidence.
Such outcomes can be frustrating, as they can leave questions about which therapeutic regimens patients should receive and the symptoms for which they should be monitored. The good news is that, as we learn more about VUS, many will be reclassified as either benign or pathogenic, opening the door to additional diagnoses.
But exactly how is a VUS transformed into an actionable mutation? How can allergists/immunologists and other clinicians contribute to the process, and what can we do in the clinic for our patients who receive VUS results?
This article, the second in a 2-part series on diagnosing rare immune disorders through genetic testing, will provide guidance around these issues.
Understanding VUS
To diagnose a disease like APDS, a progressive, underdiagnosed primary immunodeficiency, physicians must match a patient’s clinical phenotype with a pathogenic genetic variant.7 When genetic testing brings back a VUS finding, an important piece of the puzzle is missing.
In their evaluation, physicians should first consider the signs and symptoms an individual patient may be experiencing. For example, if a patient exhibits symptoms consistent with a condition such as APDS and has a VUS variant, it is likely pathogenic.
Testing a patient’s relatives for the variant will also help determine the significance of a VUS. If every family member who carries the VUS shares symptoms associated with the disease, this is also a sign that the mutation is likely pathogenic.
Doctors who uncover that kind of evidence should publish it, as sometimes that’s all it takes to convert an inconclusive finding into an actionable mutation. For instance, my team’s investigation of a family that shared a VUS within an ALPS-causing gene recently appeared in the journal Frontiers in Pediatrics. By creating a thorough medical pedigree for the family and following their genetic assays with functional immunophenotyping, we determined that the variant was likely pathogenic, and we thought it was important to share that information with the scientific community.6
Another good way to aggregate such evidence is through databases such as ClinVar. Physicians and researchers can submit VUS results to this freely accessible public archive, along with their hypotheses about the clinical significance of the variants. Also, key in solving medical mysteries is the Genome Aggregation Database (gnomAD), an open public archive of information about the genomic and exomic variations in healthy individuals.
To go a step further, a panel of experts was recently charged with assessing the role of every known variant based on functional and genetic data from ClinVar, gnomAD, and the literature. The Variant Curation Expert Panel will publish its findings in ClinVar and ClinGen’s publicly available Evidence Repository.8
Unfortunately, some mutations remain inconclusive even after an investigation, and in those cases, complementary functional assays can help physicians establish diagnoses. In conditions like APDS, in which cell biology is characterized by abnormal PI3K/Akt signaling—in other words, PTEN deficiency — clinicians can gather convincing evidence by confirming overactivation of the PI3K/Akt pathway. This strategy should soon be widely available through assays that will analyze phosphorylation levels of downstream proteins such as Akt, FOXO1, and the ribosomal protein S6.7
All of this may seem complex—even to allergists/immunologists who are typically comfortable with genetic testing. That’s why I’ve collaborated with colleagues in Florida to launch Ask the Immunologist, a quarterly series of mini-symposia designed to save patients’ lives by ensuring that their physicians can reliably distinguish rare diseases from common complaints.
By presenting case studies, project leaders highlight the role of genetic testing in the diagnosis of immune disorders, along with its challenges.
Lessons From 1 Family’s VUS Journey
My team often handles such cases, including a recent one that demonstrated the complexities surrounding a VUS finding.
Presented with 3 siblings suspected of having APDS, we conducted genetic testing that revealed a shared VUS. A frameshift variant, it was in a regulatory unit at position 1083 in the PIK3R1 protein but was truncated at the end. While the family members had a clinical history consistent with a PI3K deficiency, their immunological phenotype appeared less dramatic than full-blown APDS.
In genetic testing reports, the shared variant was deemed likely pathogenic, as it was expected to result in protein truncation or nonsense-mediated decay in a gene for which loss of function is a known mechanism of disease.
However, questions about its relationship to APDS lingered. Most importantly, while all 3 siblings had symptoms consistent with APDS, only 2 harbored the VUS. Furthermore, in reviews of ClinVar and gnomAD, we found that the variation had never been reported in either healthy or sick populations, so nothing was known about its significance.
Currently, without sufficient evidence that these siblings have APDS, the next step in our investigation will be to seek confirmation of an overactive PI3K/Akt pathway by conducting phosphorylation analysis.
This case serves as a reminder that physicians cannot decisively diagnose an immunodeficiency without first confirming the presence of a pathogenic variation. Fortunately, there’s an easy and dependable way to achieve that, regardless of a physician’s grasp of genetic intricacies.
The rule of thumb is simple: We must take our cues from genetic testing reports, basing our diagnostic decisions strictly on variants categorized as either pathogenic or benign. While it can be tempting to make assumptions about inconclusive findings, VUS should be regarded as clues in a larger investigation until their significance has been proven.
With this guideline in mind, every physician can support the accurate diagnosis and appropriate treatment of rare immunodeficiencies—as well as our growing understanding of the genetic drivers of disease.
Jolan Eszter Walter, MD, PhD, is an international expert in primary immunodeficiencies and immune dysregulation who serves as division chief of the University of South Florida and Johns Hopkins All Children’s Pediatric Allergy & Immunology Programs. Her research is focused on better understanding diseases associated with the under- and overactive immune system and defining strategies for the early detection of susceptible individuals and novel approaches for precision treatment. This article was written for educational purposes, and the views expressed herein are solely those of the author. Editorial support was provided by a third party and paid for by Pharming Healthcare, Inc. No financial compensation or incentives were provided.
References
- Angulo I, Vadas O, Garçon F, et al. Phosphoinositide 3-kinase δ gene mutation predisposes to respiratory infection and airway damage. Science. 2013;342(6160):866-871. doi:10.1126/science.1243292
- Lucas CL, Kuehn HS, Zhao F, et al. Dominant-activating germline mutations in the gene encoding the PI(3)K catalytic subunit p110δ result in T cell senescence and human immunodeficiency. Nat Immunol. 2014;15(1):88-97. doi:10.1038/ni.2771
- Deau MC, Heurtier L, Frange P, et al. A human immunodeficiency caused by mutations in the PIK3R1 gene [published correction appears in J Clin Invest. 2015 Apr;125(4):1764-5]. J Clin Invest. 2014;124(9):3923-3928. doi:10.1172/JCI75746
- Lucas CL, Zhang Y, Venida A, et al. Heterozygous splice mutation in PIK3R1 causes human immunodeficiency with lymphoproliferation due to dominant activation of PI3K. J Exp Med. 2014;211(13):2537-2547. doi:10.1084/jem.20141759
- Jamee M, et al. Clinical, immunological, and genetic features in patients with activated PI3Kδ syndrome (APDS): a systematic review. Clin Rev Allergy Immunol. 2020;59(3):323-333. doi:10.1007/s12016-019-08738-9
- Gaefke CL, Metts J, Imanirad D, et al. Case report: a novel pathogenic missense mutation in FAS: a multi-generational case series of autoimmune lymphoproliferative syndrome. Front Pediatr. 2021;9:624116. doi:10.3389/fped.2021.624116
- Vanselow S, Wahn V, Schuetz C. Activated PI3Kδ syndrome – reviewing challenges in diagnosis and treatment. Front Immunol. 2023;14:1208567. doi:10.3389/fimmu.2023.1208567
- ClinGen general sequence variant curation process standard operating procedure, version 3.2. Published October 2022. Accessed January 22, 2025.
