Germline mutations in genes encoding proteins of the RAS/MAPK signaling pathway give rise to a family of clinically related disorders, collectively known as RASopathies. Among these, cardiofaciocutaneous syndrome (CFCS; OMIM #115150, #615278, #615279 and #615280) is a rare autosomal dominant disease that affects 1/800,000 newborns (1) and it is caused by mutations in BRAF, MAP2K1, MAP2K2, KRAS and YWHAZ genes (2). CFCS is characterized by a distinctive combination of dysmorphic craniofacial features, heart and skin abnormalities, gastrointestinal dysfunction, neurocognitive delay and epilepsy (3, 4).

Inborn errors of immunity (IEI) are heterogeneous conditions that share increased susceptibility to infections, autoimmunity, allergy, autoinflammation and predisposition to malignancy (5, 6). Syndromic immunodeficiencies are a subgroup of IEI characterized by involvement of different organ systems simultaneously (7).

Recently, case reports have described the immunological phenotype of CFCS, reporting antibody deficiency in patients affected by MAP2K1 and MAP2K2 mutations (8–10). To date, there are no studies that precisely portray the immunological phenotype of CFCS, its natural history, and treatment for immunological defects.

Here we report the results of a multicenter retrospective observational study on the immunological characterization of patients with clinical and molecular confirmed diagnosis of CFCS. We aim to investigate clinical and immunological features, including rare, severe or novel phenotypes, gather data on employed treatments, and identify correlations with non-immunological traits.

One author (C.E.) carried out a systematic review of all studies published as original articles up to May 31, 2024, in PubMed database. “Cardiofaciocutaneous syndrome”. Languages included were English, Spanish, and French. The corresponding authors of all relevant publications and referring physicians were contacted to obtain updated clinical information on the reported cases. To be included in our study, the criteria were: clinical diagnosis of CFCS, pathogenic or variants of unknown significance in CFCS genes (BRAF, MAP2K1, MAP2K2, KRAS, YWHAZ), completion of the Case Report Form (CRF) and provided written informed consent either from the participant, parents or legal guardians.

Data was obtained from the available medical records and collected using a CRF. Collected data included: demographics, genetic diagnosis, dysmorphisms and organ malformation, other features of CFCS (e.g., intellectual disability, epilepsy), immunological manifestations (increased susceptibility to infections [either severe (11) or recurrent (12)], autoimmunity, lymphoproliferation), immunological tests including complete blood count (CBC) with differential, immunoglobulin levels (IgG, IgA, IgM), lymphocyte subsets, serological response to vaccinations, serum autoantibodies, treatment and need for hospitalization. Lymphocyte subsets distribution and immunoglobulin levels were classified as normal, high or low according to published age-matched reference values (13–15).

The study was approved by the local hospital Ethical Committee (PID-GENMET) and was conducted according to the Helsinki Declaration.

CFCS is a complex multisystem disorder with variable phenotypic expression. Building on the recent evidence reporting hypogammaglobulinemia in CFCS (8–10), here we describe a large international multicenter and molecularly defined cohort, providing the most comprehensive analysis of immune abnormalities in CFCS to date. We investigated immune dysregulation, susceptibility to infections, lymphocytes’ and immunoglobulins’ alterations, and treatments in affected individuals.

Our results demonstrate that immunological alterations are common in CFCS. The immunological phenotype is highly heterogeneous, ranging from asymptomatic patients to common variable immunodeficiency-like phenotypes. CFCS should be considered a syndromic immunodeficiency. Syndromic immunodeficiencies are conditions in which immunological defects may be present only in a subset of patients (16). In recent years, there has been growing interest not only in identifying novel syndromic immunodeficiencies (17–19) but also in reassessing known syndromic conditions to determine whether immunological defects have been previously overlooked (7, 20–23).

Lymphopenia, hypogammaglobulinemia, and infections emerged as recurrent features in CFCS. Although increased infection susceptibility, mostly involving the respiratory tract, was observed, opportunistic, viral or fungal severe infections were notably absent. The frequency of infections in our CFCS cohort appears lower than in other syndromic immunodeficiencies, such as in Kabuki syndrome (KS, 69%) (21), Jacobsen syndrome (JS, 50%) (22) or Rubinstein-Taybi Syndrome (RSTS, 72%) (7). While bronchiectasis, a hallmark of primary immunodeficiencies with hypogammaglobulinemia, was not reported in our cohort, it is important to note that pulmonary function testing and chest imaging were not routinely performed. Prospective studies will be needed to define the evolution and lung sequelae of recurrent infections in CFCS patients. Additionally, craniofacial anomalies, which are characteristic of CFCS, may contribute to an increased risk of recurrent upper and lower respiratory tract infections, further complicating the immunological phenotype. Despite the relatively mild impact of immune defects, 11% of our cohort required immunodeficiency-related treatments. Thus, immune evaluations should be considered in the clinical management of CFCS patients.

AEDs were commonly administered in our cohort. Although levetiracetam (24), carbamazepine (25), valproic acid (26) and phenytoin (27) have been implicated in causing hypogammaglobulinemia, we observed no significant differences in the prevalence of hypogammaglobulinemia between AED-treated and non-treated groups.

Despite the high prevalence of skin abnormalities among CFCS patients (21/36, 58.3%), seven of whom presented with nevi, no increased risk of melanoma was observed, consistent with previous reports (2). Unlike other RASopathies, which are classified as cancer-predisposing syndromes, our CFCS cohort did not exhibit malignant lymphoproliferative disorders, further distinguishing CFCS in terms of oncological risk (2).

A noteworthy finding in our study is the high incidence (25%) of immune-mediated manifestations, which have not been previously recognized as common complication of CFCS. Although immune-mediated manifestations could point toward primary immune-regulatory disorders (28), CFCS does not exhibit concomitant autoinflammation, lymphoproliferation, malignancy, and severe atopy. This suggests that, while CFCS features immune regulatory defects, it does not fit the classic definition of a primary immune regulatory disorder.

The diverse molecular landscape of CFCS contributes to the heterogeneity of its immunological phenotype. Oncogenic BRAF and MAP2K1 mutations are functionally classified based on kinase activity, RAS-dependency and dimerization status (BRAF) (29, 30) or their dependence on phosphorylation by RAF for activation (MAP2K1) (31, 32). Germline BRAF mutations affecting the cysteine-rich domain (CRD; residues 234–280), such as the recurrent Q257R in our CFCS cohort, have been classified into CRD Classes A–C based on their ability to relieve autoinhibition and promote membrane binding. These mutations lead to enhanced RAS-dependent and -independent signaling and constitutive ERK activation (33). To date PVs or LPVs in the protein kinase domain of BRAF (F468-K601) (3) and p.Y130C/H/N variants of MAP2K1 (exon 3) have both been associated with severe epileptic phenotype (34). These genotype-phenotype correlations underscore the importance of mutation-specific analyses in CFCS to refine clinical management strategies. Our findings highlight distinct immunological signatures associated with different causative mutations, particularly between BRAF and MAP2K1 variants. BRAF mutations clustering in the CRD, particularly the Q257R variant, were predominantly associated with lymphopenia—especially T-cell lymphopenia—whereas MAP2K1 mutations, particularly Y130C variant, were frequently linked to monocytosis, reduced naïve and switched-memory B cells, and hypogammaglobulinemia, suggesting differential effects on lymphocyte development and immune cell homeostasis driven by dysregulated MAPK signaling.

The mechanism by which a subset of CFCS patients shows immune alteration remains to be elucidated. The RAS/MAPK signaling pathway is fundamental for cellular homeostasis, differentiation, proliferation, and survival. To the best of our knowledge, the role of CFCS-associated proteins in the physiology of the immune system function has not been yet characterized. CFC-causing mutations dysregulate MAPK signaling resulting in increased ERK phosphorylation (35). Similar ERK hyperactivation is observed in other RASopathies, including CBL syndrome, where monocytes exhibit ERK hyperphosphorylation (36). CBL syndrome commonly shows monocytosis, increased transitional B cells and B-cell lymphocytosis (37). Although in our cohort monocytosis was a frequent finding, we were unable to systematically investigate transitional B cells, yet B cell lymphocytosis did not appear as a common feature. In vitro studies of MAP2K1 mutations (K57M, G128V, Y130C) in neuroblastoma cell lines have demonstrated increased cell proliferation and autophagy (38). Given the tightly regulated metabolic demands of highly proliferative cells, including B cells and other hematopoietic cells, metabolic dysfunction is emerging as a key contributor to IEI (7, 19, 23). Notably, mitochondrial dysfunction, affecting oxidative phosphorylation and mitochondrial proteostasis, has been described in fibroblasts from patients with RASopathies, including CFCS (39, 40). These intriguing findings highlight the possibility that RAS/MAPK signaling dysregulation in CFCS may impact immune cell energy metabolism, contributing to the observed immunological abnormalities in CFCS’ hematopoietic cells.

Currently, no targeted therapies exist for CFCS. MEK inhibitors, including trametinib, have shown promising results in treating RASopathy-associated hypertrophic cardiomyopathy (41) and severe lymphatic anomalies (42–44). As MEK inhibitors are likely to be used in additional CFCS patients in the future, it would be valuable to conduct immunological assessments before and after treatment. Studying peripheral blood immune cells from CFCS patients undergoing MEK inhibition could provide critical insights into the role of RAS/MAPK signaling in hematopoietic cell function.

This study has several limitations. Approximately 35% of the patients were identified through literature review, leading to missing or incomplete data. Most published studies focus on pediatric populations, raising the possibility that immune system abnormalities (such as infections, immune dysregulation, and lymphoproliferation) may become more pronounced with age. Consequently, immune defects in CFCS could be underrecognized, and longitudinal studies are needed to fully assess their progression. Microbiological data, including pathogen identification and cultures during infectious episodes, were not systematically collected, limiting our ability to fully characterize the infectious burden and its etiology in CFCS patients.

Additionally, dual molecular diagnoses or multilocus genomic variations have been reported in up to 5% of patients undergoing whole exome sequencing (45) and in 10% of those screened for IEI (46). Since WES was not widely performed in our cohort, we cannot exclude the possibility that some patients carry additional pathogenic variants contributing to their immunological phenotype (47).

Until recently, the connection between immunological alterations and CFCS has been reported only in a few cases, and immunodeficiency was not listed within the main symptoms that individuals with CFCS may present. Our study establishes that CFCS is associated with variable but recurring immunological abnormalities, including lymphopenia, hypogammaglobulinemia, and increased infection susceptibility. Although immune defects in CFCS appear mild compared to other syndromic immunodeficiencies, a subset of patients requires immunodeficiency-related treatments. Our findings emphasize the need for routine immunological assessments in CFCS and highlight the potential role of RAS/MAPK signaling in immune system regulation. Future studies should focus on the metabolic and signaling pathways underlying CFCS-associated immune dysfunction, as well as the impact of MEK inhibitors on immune homeostasis.