*4.2.1 STAT1 phosphorylation for the diagnosis of chronic mucocutaneous candidiasis*

STAT1 is a transcription factor which in humans can be activated by several ligands such as interferon alpha (IFNα), interferon gamma (IFNγ), epidermal growth factor (EGF), platelet derived growth factor (PDGF) or interleukin 6 (IL-6). Mutations in the STAT1 molecule can be gain of function (GOF) or loss of function (LOF). Both of them can cause PID with different phenotypes and symptoms [39].

**STAT1 GOF dominant mutations** were first discovered in patients with chronic mucocutaneous candidiasis (CMC). This disease typically shows symptoms such as persistent infections of the skin, oral/genital mucosa and nails caused mainly by *Candida albicans*. CMC may very often result from primary immunodeficiency. To assess the presence of a STAT1 GOF defect, isolated PBMCs are stimulated *in vitro* with INFγ. In order to know if there is a gain of function, we perform different experimental conditions, such as unstimulated, stimulated with INFγ, and with INFγ plus *Staurosporine*. *Staurosporine* is an inhibitor for Ca2+/calmodulin-dependent protein kinase II inhibiting STAT1 phosphorylation [40]. The use of *Staurosporine* is justified to clarify the difference between control subjects (STAT1 WT alleles) and the CMC patients (STAT1 GOF alleles). STAT1 phosphorylation is assessed in CD14+ monocytes [41]. In a healthy donor, we can observe that STAT1 phosphorylation is reduced with the addition of Staurosporine, while in a patient with STAT1 GOF, phosphorylation is maintained despite the use of Staurosporine (**Figure 8**) [42].

Regarding **STAT1 LOF autosomic recessive mutations**, there are two genetic deficiencies that compromise the response to interferons type I and III. On the one hand, there is autosomal recessive deficiency (RA), partial or even complete STAT1. These types of defects are diagnosed in patients with an increase in intracellular or viral bacterial infections, with impaired responses to IFN α, β, γ, and IL27. In complete recessive form, there is a very low response to anti-viral and antimycotical treatments. On the other hand, partial STAT1 deficiency can also be autosomal dominant (AD); phenotypically causes impaired IFNγ responses and patients suffer with selective intracellular bacterial diseases such as Mendelian susceptibility to mycobacterial diseases (MSMD). To assess if there is a STAT1 LOF, isolated PBMCs

**Figure 8.**

*The STAT1 phosphorylation and de-phosphorylation study for the diagnosis of function gain mutations (GOF) is evaluated in CD14+ monocytes. In a healthy donor (left), we can see that STAT1 phosphorylation is reduced with the addition of staurosporine (gray) while in the patient (right), phosphorylation is maintained despite the use of staurosporine (black: no staurosporine, white: control of isotypes).*

are stimulated with INFγ and phosphorylation is assessed in CD14+ monocytes. In these cases, STAT1 phosphorylation in monocytes is clearly diminished comparing with a healthy donor [43].

See STAT phosphorylation staining protocol in Section 4.2.
