Low IgE Is a Useful Tool to Identify STAT3 Gain-of-Function Mutations.
Germline gain-of-function (GoF)  mutations in signal transducer and activator of transcription 3 (STAT3)  were recently identified as the cause of early-onset autoimmune disease (MIM: 615952). This rare monogenic autoimmune disorder commonly encompasses autoimmune hematological disorders (identified in 70% of patients), recurrent infections (60%), and autoimmune enteropathies (50%) (1-3). The phenotype observed contrasts to that of patients with loss-of-function (LoF) STAT3 mutations causing hyper IgE syndrome (Job syndrome), typically characterized by increased immunoglobulin E (IgE), recurrent infections, and eczema (4). Identifying patients with STAT3 GoF mutations informs families and clinicians of prognosis and facilitates personalized treatment.
There is substantial clinical variability between patients with GoF STAT3 mutations and also overlap in phenotype with other monogenic autoimmune disorders such as immunodysregulation polyendocrinopathy enteropathy X-linked (IPEX) syndrome (MIM: 304790) resulting from hemizygous loss-of-function forkhead box P3 (FOXP3) mutations, and also early-onset polygenic autoimmune disease. Distinguishing the specific genetic etiology using clinical characteristics alone is not possible and genetic testing, often performed sequentially until a pathogenic mutation is found, is essential for accurate diagnosis.
Genetic testing for STAT3 is expensive and available only in a limited number of specialist centers. An inexpensive and widely available test to identify patients most likely to harbor GoF STAT3 mutations is desirable as it will prevent inappropriate genetic testing. We assessed whether IgE would be a useful tool to aid in the identification of patients suitable for STAT3 sequencing.
Total serum IgE concentration was measured by using the ImmunoCAP 1000 instrument (Phadia) for both healthy reference samples (n = 1510) and STAT3 patient samples (n = 6). Calibration was performed with the 2nd WHO International Reference Preparation of Human Serum Immunoglobulin E (IgE) (NIBSC code 75/502) (5). Statistical analysis was performed in Stata[R]; specificity, sensitivity and binomial confidence intervals were calculated. The age of the patients with STAT3 GoF mutations ranged from 1 to 9 years, and the median time from the first clinical symptom to IgE testing was 4 years.
Immunological assessment of individuals with STAT3 GoF mutations identified total serum IgE below the lower reference limit of age-matched controls in all 6 cases, (<2 kU/L, range 0.7-2 kU/L; see Fig. 1) (1). Using 2 kU/L as a cutoff for the assay for identification against all age groups gave a sensitivity and specificity of 100% (95% CI, 54. -100) and 97.2% (95% CI, 96.2-97.9), respectively.
We have demonstrated that serum IgE, an inexpensive and widely available diagnostic test, is likely to be very useful to facilitate identifying patients suitable for STAT3 sequencing, and a 2-kU/L cutoff gives high sensitivity and specificity. The role we propose for IgE measurement is in patients with multisystem early-childhood autoimmune disease in whom STAT3 GoF mutations are being considered. This is likely to be particularly helpful in distinguishing from multiple autoimmunity resulting from hemizygous FOXP3 mutations as more than 90% of patients with IPEX syndrome have increased serum IgE. In this scenario, raised IgE would be helpful to rule out STAT3 GoF mutations, reducing total genetic testing expenditure. In addition, despite recent advances in sequencing technologies, identifying pathogenic mutations from benign variants remains a challenge. Measuring IgE could be a tool to aid molecular geneticists in classifying STAT3 variants found by DNA sequencing; serum IgE concentration below 2 kU/L supports pathogenic GoF.
We have assessed IgE in a small number of patients with GoF STAT3 mutations (n = 6) and assessment in further patients is required to increase confidence in the diagnostic utility, but it is notable that all 6 patients had IgE concentrations below the 1st centile of the reference population. Two previous case series of patients with STAT3 GoF mutations measured IgE in a total of 9 additional individuals (4 of those previously reported are included in this study (1, 2)). One described 2 additional individuals with serum IgE below 2 kU/L (2), supporting our findings. The other reported more variability in IgE than we have observed (0.1-58.5 mg/dL; n = 7) (3), but we were unable to ascertain reference interval, testing platform or comparable units to our assay for these individuals and therefore, unable to make a direct comparison to our findings (3). Apotential limitation of the diagnostic accuracy of IgE to identify patients with STAT3 GoF mutations is that an allergic response or parasitic infection may increase serum IgE. Given that allergies are relatively common (for example, allergic rhinitis is present in >20% of the European population) this is an important consideration as this may decrease the performance of the test in these patients. In conclusion, total serum IgE concentration is an inexpensive and robust tool for determining which patients with multiple early-onset autoimmunity are likely to have a STAT3 GoF mutation. In combination with the identification of clinical features of early onset autoimmune disease, IgE concentrations could be assayed locally to facilitate appropriate, cost-efficient, and rapid genetic testing.
Further work is warranted to determine IgE in other subtypes of monogenic autoimmunity.
Author Contributions: AH authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.
Authors' Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclosure form. Disclosures and/or potential conflicts of interest:
Employment or Leadership: None declared.
Consultant or Advisory Role: None declared.
Stock Ownership: None declared.
Honoraria: None declared.
Research Funding: The Wellcome Trust. S. E. Flanagan, Sir Henry Dale Fellowship jointly funded by the Wellcome Trust and the Royal Society (grant number 105636/Z/ 14/Z); A. T. Hattersley, Wellcome Trust Senior Investigator Grant; T.J. McDonald, NIHR Chief Scientific Officer fellowship. Expert Testimony: None declared. Patents: None declared.
Acknowledgments: The authors thank Dr. T. Laver and Dr. E. De Franco for their useful comments and suggestions, and Dr. B. Shields for assistance with statistical analyses.
(1.) Flanagan SE, Haapaniemi E, Russell MA, Caswell R, Lango Allen H, De Franco E, et al. Activating germline mutations in STAT3 cause early-onset multi-organ autoimmune disease. Nat Genet 2014; 46:812-4.
(2.) Haapaniemi EM, Kaustio M, Rajala HL, van Adrichem AJ, Kainulainen L, GlumoffV, et al. Autoimmunity, hypogammaglobulinemia, lymphoproliferation, and mycobacterial disease in patients with activating mutations in STAT3. Blood 2015; 125:639-48.
(3.) Milner JD, Vogel TP, Forbes L, Ma CA, Stray-Pedersen A, Niemela JE, et al. Early-onset lymphoproliferation and autoimmunity caused by germline STAT3 gain-of-function mutations. Blood 2015; 125:591-9.
(4.) Mogensen TH. STAT3 and the hyper-IgE syndrome: clinical presentation, genetic origin, pathogenesis, novel findings and remaining uncertainties. JAKSTAT 2013; 2:e23435.
(5.) Martins TB, Bandhauer ME, Bunker AM, Roberts WL, Hill HR. New childhood and adult reference intervals for total IgE. J Allergy Clin Immunol 2014; 133:589-91.
 Nonstandard abbreviations: GoF, gain-of-function; LoF, loss-of-function; IgE, immunoglobulin E; IPEX, immunodysregulation polyendocrinopathy enteropathy X-linked syndrome.
 Human genes: STAT3, signal transducer and activator of transcription 3; FOXP3, forkhead box P3.
Matthew B. Johnson 
Sarah E. Flanagan 
Thomas B. Martins 
Harry R. Hill 
Andrew T. Hattersley 
Timothy J. McDonald [3,5] *
 Institute of Biomedical and Clinical Science University of Exeter Medical School, Exeter, Devon, UK
 Department of Pathology the ARUP Institute for Clinical and Experimental Pathology University of Utah, Salt Lake City, UT
 Molecular Genetics and Blood Sciences Royal Devon and Exeter NHS Foundation Trust, Exeter, UK
* Address correspondence to this author at: Molecular Genetics & Blood Sciences Royal Devon and Exeter NHS Foundation Trust Exeter, UK, EX2 5AD E-mail Timothy.email@example.com
Previously published online at DOI: 10.1373/clinchem.2016.263624
Caption: Fig. 1. Boxplot of serum IgE concentrations in healthy controls and patients with STAT3 GoF mutations. P value determined using the Mann-Whitney test.
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|Title Annotation:||Letters to the Editor|
|Author:||Johnson, Matthew B.; Flanagan, Sarah E.; Martins, Thomas B.; Hill, Harry R.; Hattersley, Andrew T.;|
|Article Type:||Letter to the editor|
|Date:||Nov 1, 2016|
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