Background/Purpose: A host of primary immunodeficiencies, such as autoimmune polyendocrinopathy, candidiasis, ectodermal dysplasia (APECED), immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX), and STAT5b insufficiency can present as immune dysregulation syndromes. However, in many patients the molecular basis of these abnormalities remains uncharacterized. To investigate potential genetic and molecular links between PID, growth failure and systemic autoimmunity, we recruited a series of siblings with immunodeficiency, short stature, polyendocrinopathy and kidney disease. Since our patients feature growth hormone resistance, PID and systemic autoimmunity, we hypothesized that a functional defect in the signal transducer activator of transcription 5b (STAT5b) would underlie the phenotype.

Methods: Clinical data was extracted from the patients’ medical record, patients were evaluated, and skin and blood samples were analyzed in the laboratory. PBMC and dermal fibroblast-derived gDNA was used for targeted gene analysis of known PID-causing genes. Additionally, whole genome sequencing (WGS) was performed on the parents, 3 affected sons, 3 unaffected sons and 2 unaffected sisters. Mutation search were performed with a combination of scripts developed in house and expert review. Cellular phenotyping by flow cytometry, STAT phosphorylation, T-cell proliferation in response to IL-2 and IL-7 stimulation were performed on affected individuals PBMC.

Results: The three affected individuals have a phenotype characterized by short stature with decreased IGF-1 and IGFBP-3 levels, PID with abnormal T-, B- and NK- cell subpopulations, polyglandular endocrinopathy and multisystem manifestations of immune dysregulation. Genetic analysis of candidate genes such as STAT5a/b and JAK-1 revealed no defect. Initial mutation searches were focused on rare protein coding changes that are consistent with X-linked, autosomal recessive or de novo inheritance models, however no probable candidate mutations were identified. Analysis of CNV data and haplotype analysis on X chromosome also came out negative. Linkage analysis identified 4 regions in the genome that have the maximum LOD score achievable for the given pedigree. One of the candidate regions on chr2 harbors a non-coding RNA gene (RNU4ATAC) that has been recently published to cause Roifman Syndrome. Our patients bear clinical and phenotypic similarities to those with Roifman Syndrome, suggesting RNU4ATAC as a likely candidate gene. We found two rare mutations in this gene that segregated with the disease: c.[13C>T] and c.[116A>C]. The first one has been reported as pathogenic in Roifman Syndrome. The second one is extremely rare (absent in public databases and more than 3700 internal WGS controls) residing in a gene region likely to be important for function. Sequential flow cytometry studies from three affected individuals in the same kindred revealed progressive B cell depletion and low numbers of T regulatory cells. We detected constitutive STAT5 phosphorylation in ex vivo CD4+ T cells, however, T cell blasts failed to proliferate in response to IL-2 following mitogen stimulation due to impaired upregulation of the high affinity IL-2 receptor. Therefore, U4ATAC may regulate immune cell functions such as T cell activation and the induction of T regulatory cells. Ongoing work in this area involves a combination of molecular and cellular approaches to determine whether U4ATACcontrols minor splicing of genes important in activated T cells.

Conclusion: Using WGS and mutation analysis, we have found the likely disease-causing mutations in patients with clinical features consistent with Roifman syndrome. Furthermore, our work extends the spectrum of Roifman Syndrome to include immune dysregulation. RNU4ATAC plays an essential role in splicing minor introns found in about 800 human genes. Further work may provide novel insights into the molecular pathways that link the development and function of the musculoskeletal, endocrine and immune systems with RNA splicing

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/u4atac-mutation-is-associated-with-an-immune-dysregulation-syndrome-characterized-by-primary-immunodeficiency-short-stature-and-polyglandular-endocrinopathy/