Expanding the Nude SCID/CID Phenotype Associated with FOXN1 Homozygous, Compound Heterozygous, or Heterozygous Mutations

Nude severe combined immunodeficiency (SCID) syndrome is an autosomal recessive disorder characterized by congenital alopecia, nail dystrophy, and athymia. It was described for the first time in 1996 as the human equivalent of the well-known nude murine phenotype [1]. The phenotype arises from the biallelic loss of function mutations of the FOXN1 gene, a member of the forkhead box gene family that includes a diverse group of “winged helix” transcription factors implicated in a variety of cellular processes: development, metabolism, cancer, and aging [2‐4]. During postnatal life, FOXN1 is expressed in thymic stromal and skin cells, where it is necessary for the normal development, function, and maintenance of hair follicles and thymic epithelial cells (TECs) [5‐7]. After the description of the first two cases, only few patients have been described in almost 15 years [3, 8‐12]. However, during the last few years, newborn screening (NBS) and next-generation sequencing (NGS) led to the identification of an increased number of patients, suggesting that the actual incidence of the disease is higher than previously thought and many patients die before receiving a diagnosis [13‐15]. In contrast to most the other forms of SCID, nude SCID is not related to a defect in the hematopoietic stem cells (HSC) but to a defect of the thymic epithelial stroma and this makes the definitive treatment of these conditions more complicated [16].

As for other inborn errors of immunity (IEIs), NGS allowed the diagnosis also in patients presenting with atypical phenotypes, broadening the spectrum of clinical manifestations related to different FOXN1 mutations [17, 18]. In a recent study, we described a cohort of subjects found positive on NBS in which NGS revealed heterozygous mutations in the FOXN1 gene [17]. All the pediatric patients showed lymphopenia with a tendency to resolution in older ages and about half had recurrent upper respiratory tract infections. In a few cases, the phenotype was more severe, characterized by chronic or severe invasive opportunistic or viral infections, suggesting a more severe dysfunction of FOXN1.

Signs of SCID were present at onset in all homozygous, in 1 compound heterozygous, and in 4 heterozygous patients. Lung infection was the most common infectious manifestation, observed in 12/18 patients. Half of the patients presented with signs of OS. The study of lymphocyte subpopulations revealed a T-B+NK+ phenotype in most of the cases. In a few cases, T cell levels were low-to-normal. As expected, OS was associated with higher CD3 levels that were sometimes normal. However, naïve T cells and RTEs and TRECs were very low or absent in all cases tested. Low B cell levels were detected in one patient and in a second patient marked lymphopenia resulted in T-B-NK-phenotype.

In homozygous patients, alopecia and nail dystrophy, which are usually considered the hallmarks of the syndrome, were not present in all the subjects, even though nail dystrophy may be difficult to identify. In a recent paper, Du et al. described 2 patients with FOXN1 compound heterozygous mutations who only had selective thymic hypoplasia [18]. By reproducing the mutations in mouse models generated through CRISPR-Cas9 technology, the authors confirmed that different mutations may play distinct roles in TECs and keratinocytes. In particular, they identified a region at the COOH-terminal end of the DNA binding domain that uncouples TEC development and keratinocyte differentiation [18]. In our cohort, 2 homozygous patients did not show alopecia at birth. In both patients, the mutations (S188fs and V294I) were located in the forkhead domain. However, in contrast to the 2 patients described by Du et al., in our study, the first patient developed alopecia during the follow-up in association with erythroderma and the second one had a sibling with alopecia, carrying the same mutation. In a third patient born with congenital alopecia, spontaneous hair regrowth was observed starting from 3 years of age. This patient carries 2 compound heterozygous mutations, namely a stop codon mutation (C82X) in the N-terminal domain and a missense mutation (P350L) in the forkhead domain. Alopecia was absent in 1 out of 5 members of the heterozygous family and only involved eyebrows in the remaining 4 heterozygous patients. Notably, the mutation identified in this family is very close to the mutation reported by Du et al. in the transactivation (TA) domain [18]. These observations suggest that FOXN1 activity in keratinocytes might be only partially impaired in some cases and other still unidentified factors might also play a role in regulating its activity. Alternatively, a differential dose dependency in TEC and keratinocytes with respect to FOXN1 activity may explain why certain mutations only affect TEC development. The absence of the classical hallmarks of the disease associated with the finding of normal or only slightly reduced lymphocyte count may delay the diagnosis, especially in a non-specialist setting, where the naïve compartment is not routinely assessed. Increased IgE levels and eosinophilia, also typically observed in OS, in association with erythroderma and chronic diarrhea, may also be misleading and delay the diagnosis, as observed in P6 [13]. These observations further support the importance of NBS in the timely identification of the different forms of SCID.

Thanks to the recent introduction of the NBS in combination with NGS it has been possible to unravel novel important evidence on the role of FOXN1 in human genetics. In a recent paper, we described a cohort of 21 children, identified through NBS in which NGS led to the identification of heterozygous pathogenic variants in the FOXN1 gene [17, 24, 25]. Most of the children showed nail dystrophy and some of them also experienced recurrent upper respiratory infections. Respiratory infections were rarer in adult heterozygous patients, who also showed normalization of CD4⁺ T cell levels. In a mouse model heterozygous for Foxn1 mutation, we also showed that Foxn1 plays a key role in the recruitment of the early thymic precursors and that this process is also affected by Foxn1 haploinsufficiency. In two brothers, carrying a heterozygous deletion in the C-terminal domain (P465Rfs*82) leading to a premature stop codon, the clinical phenotype was more severe, characterized by recurrent opportunistic infections and required HSCT in one patient [17]. The clinical phenotype was more similar to that observed in patients carrying homozygous mutations. These 2 patients and their younger sibling were better analyzed in our study and the phenotype was additionally evaluated in other family members carrying the same FOXN1 variant. In particular, the father experienced severe infections requiring hospitalization and admission to the intensive care unit in the first few years of life. Differently, the paternal grandmother, who also carried the mutation, never experienced severe infections but developed type 1 diabetes mellitus and psoriasis. The three of them had eyebrow alopecia. Interestingly, the mutation identified is very close to the mutation reported by Du et al. in the TA domain [18]. In the patient described by Du et al., the mutation in the TA domain was associated with a polymorphism with no functional relevance on the other allele, suggesting that this patient was also heterozygous [18]. Even if we were not able to characterize in vitro the variant identified, the severity of the clinical phenotype suggests that FOXN1 function was more severely impaired in these patients compared to the other heterozygous subjects. Different mechanisms may explain this finding. This mutation may potentially lead to a mechanism of negative dominance. However, in our report, the clinical phenotype in the grandmother, carrying the same mutation identified in the 3 siblings, was not as severe. The observation that all the children within this family were symptomatic, while the adults were not, indicates that the requirement of FOXN1 varies with the age, as already described in heterozygous mice, whose immunological alterations improve during adulthood. Another explanation may be the presence of additional mutations in noncoding regions of FOXN1. Recently, Larsen et al. identified intronic regulatory elements that play a key role in tissue-specific expression of Foxn1 in TECs [26]. In particular, the authors identified a highly conserved 1.6-kb region in the first intron of Foxn1 whose deletion leads to a complete abrogation of Foxn1 expression and thymus development. Of note, keratinocyte differentiation is not affected by deleting this region [26]. More studies will be necessary to confirm these hypotheses. Interestingly, one of the 2 brothers developed EBV-related diffuse large B cell lymphoma, and EBV-related B cell lymphoma was the presenting manifestation in P9 and P11. EBV-related B cell lymphomas represent a well-known complication in secondary immunodeficiencies and IEIs [27]. Different IEIs affecting NK and T cell function are associated with the development of chronic EBV infections, eventually leading to lymphoma [28, 29]. In this study, 3 patients, carrying homozygous, compound heterozygous, or heterozygous mutations developed EBV-related lymphomas, suggesting that T cell lymphopenia in FOXN1-deficient patients impairs the immunological control of EBV infection. In P9, uncontrolled EBV infection also led to the development of infiltrative lung disease. P9, carrying two compound heterozygous mutations, did not show any signs of SCID at the onset. His phenotype was characterized by late-onset chronic EBV infection, leading to the development of B cell lymphoma and lung infiltration. It should be noted that in this patient, a stop mutation was associated with a missense mutation, differently from P8, presenting with a typical Nude SCID phenotype, and carrying 2 different stop mutations.

A spectrum of clinical manifestations may be associated with different mutations, and the severity of the clinical phenotype likely depends on the amount of residual activity of the gene product, as previously observed for other SCID-related genes [30]. Similarly to what recently observed by Du et al. in a patient carrying a heterozygous mutation affecting the TA domain [18], in a family carrying a heterozygous mutation, we observed an unexpectedly severe phenotype, characterized by CID in 4 out of 5 patients, suggesting that heterozygous mutations may also present with severe clinical phenotypes. A negative dominant mechanism or the presence of additional alterations in noncoding regions may explain the different severity compared to other heterozygous mutations. More studies are necessary to better characterize the effect of the different mutations and to define a clear-cut clinical strategy for delineating genotype-phenotype relationships to guide clinicians towards the best therapeutic choice. Moreover, as for other types of atypical combined immunodeficiencies, further studies are necessary to define the best therapeutic approach. In fact, both thymus transplant and HSCT may result in the development of severe complications [31‐34]. Though numbers are too small to make a definitive conclusion, the survival after thymus transplantation was better than after HSCT and in three patients, there was good evidence of naïve T cell production. As already reported, homozygous mutation leads to a SCID phenotype that may befit thymus transplantation since HSCT may be ineffective. As for compound heterozygous mutations, although a milder phenotype has been reported, symptoms may be severe as well and must also be considered the risk of lymphoma. Thus, a decision on the therapeutic strategy should be taken on an individual case basis. Differently, the phenotype associated with heterozygous mutations is usually absent or very mild and no therapeutic intervention is required. Our recent study suggests that lymphopenia and susceptibility to infections in heterozygous patients tends to improve with age. This improvement is not dependent on a recovery of the thymus function and the defect of the CD8 compartment usually persists also in the adults [17]. Studies are needed to define whether the phenotype in atypical heterozygous or compound heterozygous tends to improve with age and which treatment is the most indicated in these cases.