Primary immunodeficiencies (PIDs) refer to a clinically, immunologically, and genetically heterogeneous group of over 350 disorders affecting development or function of the immune system. The increasing use of next-generation sequencing (NGS) technology has greatly facilitated identification of genetic defects in PID patients in daily clinical practice. Several NGS approaches are available, from the unbiased whole exome sequencing (WES) to specific gene panels. Here, we report on a 3-year experience with clinical exome sequencing (CES) for genetic diagnosis of PIDs. We used the TruSight One sequencing panel, which includes 4,813 disease-associated genes, in 61 unrelated patients (pediatric and adults). The analysis was done in 2 steps: first, we focused on a virtual PID panel and then, we expanded the analysis to the remaining genes. A molecular diagnosis was achieved in 19 (31%) patients: 12 (20%) with mutations in genes included in the virtual PID panel and 7 (11%) with mutations in other genes. These latter cases provided interesting and somewhat unexpected findings that expand the clinical and genetic spectra of PID-related disorders, and are useful to consider in the differential diagnosis. We also discuss 5 patients (8%) with incomplete genotypes or variants of uncertain significance. Finally, we address the limitations of CES exemplified by 7 patients (11%) with negative results on CES who were later diagnosed by other approaches (more specific PID panels, WES, and comparative genomic hybridization array). In summary, the genetic diagnosis rate using CES was 31% (including a description of 12 novel mutations), which rose to 42% after including diagnoses achieved by later use of other techniques. The description of patients with mutations in genes not included in the PID classification illustrates the heterogeneity and complexity of PID-related disorders.

Purpose STAT1 gain-of-function (GOF) and dominant-negative (DN) STAT3 syndromes share clinical manifestations including infectious and inflammatory manifestations. Targeted treatment with Janus-kinase (JAK) inhibitors shows promising results in treating STAT1 GOF-associated symptoms while management of DN STAT3 patients has been largely supportive. We here assessed the impact of ruxolitinib on the JAK-STAT1/3 pathway in DN STAT3 patients' cells. Methods Using flow cytometry, immunoblot, qPCR, and ELISA techniques, we examined the levels of basal STAT1 and phosphorylated STAT1 (pSTAT1) of cells obtained from DN STAT3, STAT1 GOF patients, and healthy donors following stimulation with type I/II interferons (IFNs) or interleukin (IL)-6. We also describe the impact of ruxolitinib on cytokineinduced STAT1 signaling in these patients. Results DN STAT3 and STAT1 GOF resulted in a similar phenotype characterized by increased STAT1 and pSTAT1 levels in response to IFNα (CD3 + cells) and IFNγ (CD14 + monocytes). STAT1-downstream gene expression and C-X-C motif chemokine 10 secretion were higher in most DN STAT3 patients upon stimulation compared to healthy controls. Ex vivo treatment with the JAK1/2-inhibitor ruxolitinib reduced cytokine responsiveness and normalized STAT1 phosphorylation in DN STAT3 and STAT1 GOF patient' cells. In addition, ex vivo treatment was effective in modulating STAT1 downstream signaling in DN STAT3 patients. Conclusion In the absence of effective targeted treatment options for AD-HIES at present, modulation of the JAK/STAT1 pathway with JAK inhibitors may be further explored particularly in those AD-HIES patients with autoimmune and/or autoinflammatory manifestations.

Chédiak-Higashi syndrome (CHS) is a rare autosomal recessive (AR) immune disorder that has usually been associated to missense, nonsense or indels mutations in the LYST gene. In this study, we describe for the first time the case of a CHS patient carrying a homozygous mutation in the LYST gene inherited as a result of a partial uniparental isodisomy (UPiD) of maternal origin. Sanger sequencing of the LYST cDNA and single nucleotide polymorphism (SNP)-arrays were performed to identify the causative mutation and to explain the molecular mechanism of inheritance, respectively. Partial-UPiD leads to a copy neutral loss of heterozygosity (CN-LOH) of the telomeric region of chromosome 1 (1q41q44), unmasking the potential effect of the mutation detected. The mutation (c.8380dupT) is an insertion located in exon 32 of the LYST gene resulting in a premature stop codon and leading to the loss of all the conserved domains at the C-terminal of the LYST protein. This would account for the severe phenotype observed. We also reviewed the only two previously reported cases of CHS as a result of a uniparental disomy. In this study, we show that the combination of different strategies, including the use of SNP-arrays, is pivotal to fine-tune the diagnosis of rare AR disorders, such as CHS. Moreover, this case highlights the relevance of uniparental disomy as a potential mechanism of CHS expression in non-consanguineous families.

Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder characterized by immunodeficiency, eczema, and thrombocytopenia with small platelets. A wide spectrum of mutations in the WiskottAldrich syndrome protein (WASP) gene have been identified as causative of the disease. In the present paper, we report on a family with a boy affected by WAS, with a splice-site mutation caused by a T to G substitution in the +2 position of intron 6 (IVS6+2T>G). Expression studies performed in COS-7 and U-937 cells showed that the mutation affected the normal splicing process. As a consequence, an abnormally long transcript of 38 nucleotides is generated. Such missplicing is probably due to the activation of a cryptic splice donor site located 38 nt downstream of exon 6. The translation of such aberrant mRNA will produce a truncated protein with a premature stop at codon 190. Thus, a novel splice-site mutation is reported in a patient with a mild WAS phenotype.

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