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Clinical and structural impact of mutations affecting the residue Phe367 of FOXP3 in patients with IPEX syndrome
Clinical Immunology 163 (2016) 60–65
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Clinical and structural impact of mutations affecting the residue Phe367 of FOXP3 in patients with IPEX syndrome Roger Colobran a,b,⁎,1, Elena Álvarez de la Campa c,1, Pere Soler-Palacín d, Andrea Martín-Nalda d, Ricardo Pujol-Borrell a,b, Xavier de la Cruz c,e, Mónica Martínez-Gallo a,b a
Immunology Division, Hospital Universitari Vall d'Hebron (HUVH). Vall d'Hebron Research Institute (VHIR), Barcelona, Catalonia, Spain Department of Cell Biology, Physiology and Immunology, Universitat Autònoma de Barcelona (UAB), Barcelona, Catalonia, Spain Research Unit in Translational Bioinformatics, Vall d'Hebron Research Institute (VHIR), Universitat Autònoma de Barcelona (UAB), Barcelona, Catalonia, Spain d Pediatric Infectious Diseases and Immunodeficiencies Unit, Hospital Universitari Vall d'Hebron (HUVH). Vall d'Hebron Research Institute (VHIR), Universitat Autònoma de Barcelona (UAB), Barcelona, Catalonia, Spain e Institució Catalana de Recerca i Estudis Avançats (ICREA), Catalonia, Spain b c
a r t i c l e
i n f o
Article history: Received 21 August 2015 Received in revised form 2 December 2015 accepted with revision 30 December 2015 Available online 31 December 2015 Keywords: FOXP3 IPEX Primary immunodeficiency Autoimmunity Mutation
a b s t r a c t Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome is a monogenic autoimmune disease characterized by early-onset life-threatening multisystemic autoimmunity. This rare hereditary disorder is caused by loss-of-function mutations in the gene encoding the forkhead box P3 (FOXP3) transcription factor, which plays a key role in the differentiation and function of CD4+ CD25+ natural regulatory T cells (Tregs), essential for the establishment and maintenance of natural tolerance. We identified a novel mutation in the FOXP3 gene affecting the Phe367 residue of the protein (F367V) in a family with three male siblings affected by IPEX. Two other mutations affecting the FOXP3 Phe367 residue (F367L and F367C) have been described previously. This unique situation of three mutations affecting the same residue in FOXP3 led us to study the molecular impact of these mutations on the structure of FOXP3 protein. Structure analysis showed that Phe367 is involved in a rich interaction network related to both monomer and dimer structure stabilization, and is crucial for FOXP3 regulatory activity. The relevance of this location is confirmed by the results of SIFT and PolyPhen-2 pathogenicity predictions for F367V mutation. In summary, as assessment of the pathogenicity of a novel mutation is crucial to achieve a proper molecular diagnosis, we analysed the impact of mutations affecting the Phe367 residue using a combined approach that provides a mechanistic view of their pathogenic effect. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome (OMIM #304790) is a rare monogenic primary immunodeficiency characterized by multiorgan autoimmunity, including severe diarrhoea due to enteropathy, chronic dermatitis, endocrinopathy (e.g., type 1 diabetes mellitus, hypothyroidism) and other organspecific diseases such as anaemia, thrombocytopenia, hepatitis, and nephritis [1]. IPEX is an X-linked recessive disorder with onset in infancy, and is often fatal by the age of 2 years if aggressive treatment is not used [2,3]. Long-term therapeutic options include immunosuppression and haematopoietic stem cell transplantation [4,5]. The immunopathogenesis of IPEX is explained by a loss of functional CD4+ CD25+ T regulatory cells (Tregs), which are critical for maintaining immune tolerance [6]. This loss is caused by mutations in the ⁎ Corresponding author at: Servei d'Immunologia, Edifici Laboratoris, Planta Baixa, Passeig Vall d'Hebron 119-129, 08035 Barcelona, Spain. E-mail address: [email protected] (R. Colobran). 1 These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.clim.2015.12.014 1521-6616/© 2015 Elsevier Inc. All rights reserved.
transcription factor, forkhead box p3 (FOXP3), the master gene for Treg differentiation [7]. More than 60 FOXP3 mutations have been identified and characterized in IPEX, each affecting Treg development or function [8]. FOXP3 deficiency results in a paucity of these cells, leading to a severe disruption of immunological tolerance and ultimately, aggressive multiorgan autoimmune disease. The FOXP3 genotype/clinical phenotype correlation in IPEX is not straightforward, but some general principles have emerged. For example, a relatively mild clinical phenotype, with compromised regulatory function but near-normal protein levels and normal Treg counts is associated with four mutation types: missense mutations and small inframe deletions that do not destroy the functional domain of any protein, and deletions and mutations in the promoter and 5′ untranslated region [9,10]. In contrast, mutations abrogating expression of functional FOXP3 (certain missense variants, as well as nonsense and frameshift mutations or splicing defects resulting in premature stop codons) tend to produce a more severe phenotype [8]. Nonetheless, disease severity is not always related to the absence of protein expression, and most individuals that carry missense mutations express the mutated protein at levels ranging from low to normal. This, together with the fact that
R. Colobran et al. / Clinical Immunology 163 (2016) 60–65
disease manifestations may differ considerably between patients with the same mutation, suggests that severity can be modulated by modifying genes affecting Treg function. As always when comparing the immune system between individuals, it should be kept in mind that phenotypic variability may also result from differences in the HLA haplotype and the lymphocyte repertoires [11]. Another factor that increases the complexity of the genotype/clinical phenotype relationship in IPEX is that other genetic defects affecting Treg function can originate an IPEX-like phenotype. These include loss-of-function mutations in CD25, STAT5B, and ITCH and gain-offunction mutations in STAT1 [12]. Furthermore, despite the progress attained in identifying the molecular basis of IPEX-like diseases, the underlying defects in a large percentage of individuals affected with these conditions remain obscure. Here, we present a study in which IPEX was suspected in three siblings, based on the clinical history of the family, starting 20 years ago. Identification of a novel mutation in the Phe367 residue of FOXP3 in the family led us to study the molecular impact of this and other mutations in the same residue on the structure of the FOXP3 protein. Functionally, this protein acts as a component of a dynamic multisubunit complex involved in histone modification and chromatin remodelling after T-cell receptor stimulation [13,14]. Its three-dimensional structure, in complex with NFAT1 (the DNA-binding domain of the nuclear factor of activated T cells-1) and a DNA fragment from interleukin-2 promoter, has been recently resolved experimentally [15]. Based on this structure, we analysed the spatial neighbourhood of Phe367 and its relationship with other FOXP3 mutations. Our results show that Phe367 is part of a rich network of hydrophobic interactions that are crucial for stabilizing both monomer and dimer structures. Many known IPEX-causing mutations are clustered in this region, suggesting a high sensitivity to sequence variations. The structural information provided here may be of value for future characterization of new mutations in this FOXP3 region. 2. Material and methods 2.1. Study sample The family reported here attended the Paediatric Infectious Diseases and Immunodeficiencies Unit of Hospital Universitari Vall d'Hebron (Barcelona, Spain). Informed consent was obtained from the family members studied, following the procedure of the Ethics Committee of Hospital Universitari Vall d'Hebron.
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3. Results 3.1. Family with suspected IPEX We report a non-consanguineous Spanish family with five siblings (three boys and two girls) from two different fathers (Fig. 1A). The family history included a maternal uncle who died due to hepatitis and diarrhoea at the age of three months. The three boys experienced earlyonset diarrhoea (clinical data in Table 1). The first boy, born in 1970, died at the age of two months because of probable infectious meningoencephalitis. He showed thymic hypoplasia, thrombocytopenia, pulmonary haemorrhage, and eosinophilic infiltrate in the gastrointestinal tract and adrenal glands. The second boy, born of a second marriage in 1986, had growth retardation, persistent diarrhoea, severe skin lesions and leucocytosis with eosinophilia in the first months of life. The infant died at the age of 13 months due to Klebsiella spp. sepsis. The third boy, born in 1988, showed growth retardation, hepatosplenomegaly, diarrhoea and severe eczema, leucocytosis with eosinophilia and had high IgE levels (N2000 kU/L). Several sepsis episodes caused by bacteria and fungi led to the child's death at the age of 13 months. Available hematologic and immunologic data of the patients are shown in Table 2. 3.2. Identification of the FOXP3 p.Phe367Val mutation Since the three affected siblings died more than 20 years ago, we were only able to trace DNA from the post mortem study of case 2. Unfortunately, the DNA was too degraded to successfully amplify all FOXP3 exons; hence, we resorted to study of the mother. Complete sequencing of the 12 exons of FOXP3 in the mother revealed a heterozygous nucleotide change (T N G) in exon 11, affecting the first position of codon 367 and leading to an amino acid change (phenylalanine to valine). This mutation had not been reported in the literature or databases, and, following the recommendations of the Human Genome Variation Society (HGVS), we named it c.1099 T N G/p.Phe367Val (Fig. 1B). To confirm that the mutation was present in at least one of the three affected siblings, we designed a specific pair of primers to amplify a short fragment of DNA containing the mutation from the sibling who had undergone post-mortem study. We successfully amplified and sequenced the FOXP3 fragment containing the mutation and confirmed that the boy had been a hemizygous carrier of the p.Phe367Val mutation. On study of the other family members, we found that the older sister was also a carrier, and genetic counselling was indicated (Fig. 1A). 3.3. Molecular impact of the F367V mutation
2.2. Mutation analysis Genomic DNA was extracted from EDTA-containing whole blood samples using a QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Polymerase chain reaction (PCR) to amplify the 12 exons of FOXP3 and their flanking regions was carried out using the primers and PCR conditions specified in Supplementary Table 1. Purified PCR products were sequenced on an ABI 3100 DNA Sequencer (Applied Biosystems, Foster, VA, USA). 2.3. Structure analyses and modelling All the structure analyses performed, such as the location of known mutations and contact computations, were done using the experimentally resolved structure of FOXP3 as reference (PDB code: 3QRF) [15]. To identify intradomain and interdomain residue-residue interactions, two residues were considered to be in contact when at least two of their atoms were located at a distance of 5 Å or less. Visual inspection to assess the impact of the mutations and creation of the structure images in the figures was done with the PyMol software package (PyMOL Molecular Graphics System, Version 1.7.4 Schrödinger, LLC).
To understand the molecular repercussions of the F367V mutation on FOXP3 function, we analysed its impact on the structure and predicted interactions of the protein. We found that the mutated residue (F367) locates within the experimentally determined structure of the DNA-binding domain (forkhead) of FOXP3, available in complex with NFAT1, the DNA-binding domain of nuclear factor of activated T cells1, and a DNA fragment from the promoter of interleukin 2 [15]. We analysed this structural information to provide a mechanistic view of the pathogenic effect of mutations at position Phe367. Subsequently, we sought additional support for this view by mapping the known FOXP3 pathological mutations to the forkhead structure [8]. 3.3.1. F367 is a key structural residue of the DNA-binding domain of FOXP3 The mutation described in this study (F367V) is not the only one occurring at position 367; two others, F367L and F367C, have been described. F367L has been reported in two separate IPEX patients: a Japanese infant with neonatal diabetes mellitus, intractable diarrhoea, liver dysfunction, thrombocytopenia, and sepsis who died at 16 weeks of age [16], and a French child with similar clinical manifestations [17] (Table 1). The group that performed the second study later reported the F367C mutation in a patient with severe protracted diarrhoea and
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Fig. 1. A. Main clinical characteristics of the three patients with IPEX and the family pedigree. B. Genetic analysis of the mother (carrier) showing the FOXP3 mutation c.1099TNG (p. Phe367Val) in heterozygous state.
protein-losing enteropathy [18] (Table 1). The description of additional mutations at F367 reinforced the idea that F367V would be pathogenic, and that position 367 is structurally and functionally critical. To test this idea, we analysed the structural environment of the wild-type residue, Phe367, using the forkhead structure in the NFAT1:FOXP3:DNA complex [15] (Fig. 2A). We found that Phe367 has an important role in both monomeric and dimeric structures (Fig. 2, B and C). Located in the H2 helix, Phe367 has 10 intra-monomer contacts of a mainly hydrophobic nature (Fig. 2B) that are an important part of the interaction network, stabilizing helices H1 and H2 in their relative orientation (Fig. 2, B and C). The significance of this contribution to the stability of the
forkhead domain is underlined by the relatively small size of the domain (87 amino acids). In accordance with this analysis, the FoldX stability computations, which provide an estimate of the change in protein stability occurring with a mutation [19], indicated that the three mutations at position 367 have a destabilizing effect, with values of 3.1 Kcal/mol (F367V), 1.7 Kcal/mol (F367L), and 4.7 Kcal/mol (F367C). In parallel, as we know that the functional form of FOXP3 is a dimer [20], we checked the location of Phe367 relative to the monomermonomer interface. We found that Phe367 contributes four hydrophobic residue-residue contacts (Fig. 2, C and D) to the forkhead functional dimer interface. This supports the idea that Phe367 has a functionally
Table 1 Clinical features of patients with mutations affecting the F367 residue of FOXP3. Reported onset F367V-P1 1 month
Endocrinopathy Enteropathy Skin Haematological manifestations anomalies
Recurrent infections
HSCT Outcome
Ref.
No
NA
No
This study
No
No
F367V-P2 1 month
No
Yes
Yes
F367V-P3 1 month
No
Yes
Yes
F367C
15
T1D
Yes
F367L
months 8 days
T1D
F367L
b6
T1D
NA
Yes. Bacteria (GNB)
No
Yes. Bacteria (Staphylococcus aureus, Enterobacter cloacae, E. coli, Acinetobacter). Fungi NA
No
No
Yes (eosinophilia) Yes (eosinophilia) NA
NA
Yes
Yes
Yes
Yes
No
Yes
Yes
No
Yes
NA
months GNB, gram-negative bacteria; HSCT, haematopoietic stem cell transplantation.
Died at 3 months Died at 13 months Died at 13 months NA Died at 4 months NA
This study This study [18] [16] [17]
R. Colobran et al. / Clinical Immunology 163 (2016) 60–65 Table 2 Hematologic and immunologic data of the IPEX patients.
Age Red blood cells (RBCs) Haemoglobin (gr/dl) Haematocrit (%) MCV (fL/red cell) Leukocytes (abs) Lymphocytes (%) Lymphocytes (abs) Monocytes (%) Monocytes (abs) Eosinophils (%) Eosinophils (abs) IgA (mg/dL) IgG (mg/dL) IgM (mg/dL) IgE (kU/L) Ratio CD4/CD8
Case 1
Case 2
Case 3
Reference values
– NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA
5 months 4.75 4 35.1 74 15,900 51 8109 4 636 10 1590 NA NA NA NA NA
11 months NA NA NA NA 35,000 28 9800 1 350 15 5250 18 407 82 N2000 2.1
3.8–4.8 10.5–13.5 33–39 77–95 5000–13,000 20–50 2300–5400 2–11 100–1000 0–5 50–250 11–153 196–1045 40–140 0–90 1.6–3.9
Abnormal values are shown in bold. MCV, mean corpuscular volume; Abs, absolute number; NA, not available.
relevant role, resulting from its contribution to the dimeric state of FOXP3, and that mutations at this location may have a disruptive effect on the dimer structure. The three mutations affecting the Phe367 residue result in a reduction in volume (F367V, −30 Å3; F367L, −11 Å3; F367C, −49 Å3), and it has been shown that interfacial cavity-creating mutations contribute to destabilize protein complexes through a combination of mechanisms, such as a hydrophobic effect or loss of van der Waals contacts [21]. In summary, structure analysis showed that Phe367 is involved in a rich interaction network related to stabilization of monomer and dimer structures. The relevance of this contribution is reinforced by the high conservation of Phe367 and its spatial neighbours on multiple sequence alignment of the FOXP3 family (Fig. 3). This is consistent with a
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pathogenic nature of the mutations under discussion, which may result from destabilization of the protein structure or direct disruption of the functional site [22,23]. 3.3.2. FOXP3 dimer interface is sensitive to sequence changes To provide further support for the results of the previous section, we mapped 29 known FOXP3 mutations [8] to the structure of the forkhead domain (Fig. 4; Supplementary Table 2). Many of them were located at the helices forming the dimer's interface (Supplementary Table 2) and involved a core of 8 aromatic residues known to stabilize the dimer [15]. Pathogenic mutations are listed for four residues of this core, and for at least one sequence neighbour of the remaining four residues (Supplementary Table 2). In addition, some of the mutated residues were within contact distance of Phe367 (Supplementary Table 2). This further supports the idea that Phe367 is part of a delicate interaction network whose integrity is fundamental for FOXP3 regulatory activity. 4. Discussion In this study, we describe the case of a family in which three members had a clinical phenotype consistent with IPEX syndrome. Sequencing of the FOXP3 gene showed a novel amino acid change (F367V) that had not been reported previously as a single-nucleotide polymorphism (SNP) or mutation. Prediction of the pathogenicity of the F367V mutation was important to establish an accurate molecular diagnosis and proper genetic counselling, as an increasing number of patients show a phenotype consistent with IPEX but do not harbour FOXP3 mutations [12]. Two other mutations affecting the same residue, F367L and F367C, have been described [16–18]. This makes F367 unique, because there are no other residues in FOXP3 where 3 different mutations have been reported in unrelated patients. This novel finding led us to analyse the impact of the mutation using an approach combining different tools. First, we studied the structural
Fig. 2. The structural environment of F367. A. Overall view of the location of Phe367 (shown using red spheres) relative to the FOXP3 forkhead (dimer, with monomers in lilac and light orange)-NFAT1 (green)-DNA (orange) complex. Relevant helices in the forkhead domains, (H1, H2 and H3) are indicated. B. Intramonomer and (C.) intermonomer contacts of residue Phe367 (shown in red). D. Detailed contact list for residue F367. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 3. Sequence conservation in the FOXP3 family. A. Local view of the multiple sequence alignment of FOXP3 in the neighbourhood of Phe367 (red arrow). Black arrows indicate the spatial residues of Phe367. B. Whole FOXP3 plot. Conservation is represented using Shannon entropy, a parameter routinely used for scoring pathogenic mutations, whose values range from 0 (complete conservation) to 4.32 (all amino acids are equiprobable at that position). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
neighbourhood of Phe367 and its relationship with known FOXP3 mutations, using the 3D FOXP3 reference structure [15]. The analysis showed that Phe367 is in a sensitive location, corresponding to the dimer interface of the FOXP3 functional form. This interface is densely affected with known pathogenic mutations [8], indicating its functional importance and suggesting a pathogenic role for F367V. To further support this conclusion, we tested the putative pathogenicity of F367V with two widely used prediction software packages, SIFT and PolyPhen-2. SIFT gave a deleterious prediction, in accordance with our structure analysis. The same happened with PolyPhen-2, once its multiple sequence alignment was corrected for the presence of a problematic sequence (see Supplementary Fig. 1). In summary, this extensive accumulation of evidence clearly indicates that the F367V mutation is pathogenic. In addition, the structure
analyses provide a relatively straightforward interpretation of the impact of the mutation, considering the functional value of the forkhead domain of FOXP3. This domain, which is the locus of Phe367, mediates DNA binding, acting as an activator/repressor [24]. FOXP3 binding requires a multimerization step, and the forkhead domain seems necessary for homo-multimerization of FOXP3 [15,25]. Previous studies have provided evidence that mutations in the forkhead domain of IPEX patients affect DNA binding [26,27]. Our work refines this view, indicating that mutations near or at the forkhead-forkhead interface may disrupt the network of interactions, impeding or hampering formation of the FOXP3-DNA complex and subsequently originating IPEX disease. This family case study illustrates the importance of reviewing clinical data in family members in whom a primary immunodeficiency is suspected. The in silico studies provided clues to evaluate the potential pathogenic effect of a genetic mutation in the absence of biologic material to study Tregs. Further descriptions of the molecular bases of primary immunodeficiencies will enable accurate preconception counselling in the relatives of children with these conditions.
Conflict of interest statement The authors declare no commercial or financial conflict of interest.
Acknowledgements
Fig. 4. Location of known FOXP3 mutated residues in the forkhead dimer. One monomer is shown in red, the other in light orange. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
The authors are thankful to the family members studied and the physicians involved in their care. We especially thank Pedro Domínguez Sampedro and Beatriz Valle del Barrio for their help in obtaining clinical data of the patients. We also acknowledge Celine Cavallo for their English language assistance. This study was funded by the Instituto de Salud Carlos III, grants PI11/1086 and PI14/00405, cofinanced by the European Regional Development Fund (ERDF), and by the Spanish Ministry of Economy and Competitiveness, grant BIO2012-40133.
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Clinical and structural impact of mutations affecting the residue Phe367 of FOXP3 in patients with IPEX syndrome Roger Colobran a,b,⁎,1, Elena Álvarez de la Campa c,1, Pere Soler-Palacín d, Andrea Martín-Nalda d, Ricardo Pujol-Borrell a,b, Xavier de la Cruz c,e, Mónica Martínez-Gallo a,b a
Immunology Division, Hospital Universitari Vall d'Hebron (HUVH). Vall d'Hebron Research Institute (VHIR), Barcelona, Catalonia, Spain Department of Cell Biology, Physiology and Immunology, Universitat Autònoma de Barcelona (UAB), Barcelona, Catalonia, Spain Research Unit in Translational Bioinformatics, Vall d'Hebron Research Institute (VHIR), Universitat Autònoma de Barcelona (UAB), Barcelona, Catalonia, Spain d Pediatric Infectious Diseases and Immunodeficiencies Unit, Hospital Universitari Vall d'Hebron (HUVH). Vall d'Hebron Research Institute (VHIR), Universitat Autònoma de Barcelona (UAB), Barcelona, Catalonia, Spain e Institució Catalana de Recerca i Estudis Avançats (ICREA), Catalonia, Spain b c
a r t i c l e
i n f o
Article history: Received 21 August 2015 Received in revised form 2 December 2015 accepted with revision 30 December 2015 Available online 31 December 2015 Keywords: FOXP3 IPEX Primary immunodeficiency Autoimmunity Mutation
a b s t r a c t Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome is a monogenic autoimmune disease characterized by early-onset life-threatening multisystemic autoimmunity. This rare hereditary disorder is caused by loss-of-function mutations in the gene encoding the forkhead box P3 (FOXP3) transcription factor, which plays a key role in the differentiation and function of CD4+ CD25+ natural regulatory T cells (Tregs), essential for the establishment and maintenance of natural tolerance. We identified a novel mutation in the FOXP3 gene affecting the Phe367 residue of the protein (F367V) in a family with three male siblings affected by IPEX. Two other mutations affecting the FOXP3 Phe367 residue (F367L and F367C) have been described previously. This unique situation of three mutations affecting the same residue in FOXP3 led us to study the molecular impact of these mutations on the structure of FOXP3 protein. Structure analysis showed that Phe367 is involved in a rich interaction network related to both monomer and dimer structure stabilization, and is crucial for FOXP3 regulatory activity. The relevance of this location is confirmed by the results of SIFT and PolyPhen-2 pathogenicity predictions for F367V mutation. In summary, as assessment of the pathogenicity of a novel mutation is crucial to achieve a proper molecular diagnosis, we analysed the impact of mutations affecting the Phe367 residue using a combined approach that provides a mechanistic view of their pathogenic effect. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome (OMIM #304790) is a rare monogenic primary immunodeficiency characterized by multiorgan autoimmunity, including severe diarrhoea due to enteropathy, chronic dermatitis, endocrinopathy (e.g., type 1 diabetes mellitus, hypothyroidism) and other organspecific diseases such as anaemia, thrombocytopenia, hepatitis, and nephritis [1]. IPEX is an X-linked recessive disorder with onset in infancy, and is often fatal by the age of 2 years if aggressive treatment is not used [2,3]. Long-term therapeutic options include immunosuppression and haematopoietic stem cell transplantation [4,5]. The immunopathogenesis of IPEX is explained by a loss of functional CD4+ CD25+ T regulatory cells (Tregs), which are critical for maintaining immune tolerance [6]. This loss is caused by mutations in the ⁎ Corresponding author at: Servei d'Immunologia, Edifici Laboratoris, Planta Baixa, Passeig Vall d'Hebron 119-129, 08035 Barcelona, Spain. E-mail address: [email protected] (R. Colobran). 1 These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.clim.2015.12.014 1521-6616/© 2015 Elsevier Inc. All rights reserved.
transcription factor, forkhead box p3 (FOXP3), the master gene for Treg differentiation [7]. More than 60 FOXP3 mutations have been identified and characterized in IPEX, each affecting Treg development or function [8]. FOXP3 deficiency results in a paucity of these cells, leading to a severe disruption of immunological tolerance and ultimately, aggressive multiorgan autoimmune disease. The FOXP3 genotype/clinical phenotype correlation in IPEX is not straightforward, but some general principles have emerged. For example, a relatively mild clinical phenotype, with compromised regulatory function but near-normal protein levels and normal Treg counts is associated with four mutation types: missense mutations and small inframe deletions that do not destroy the functional domain of any protein, and deletions and mutations in the promoter and 5′ untranslated region [9,10]. In contrast, mutations abrogating expression of functional FOXP3 (certain missense variants, as well as nonsense and frameshift mutations or splicing defects resulting in premature stop codons) tend to produce a more severe phenotype [8]. Nonetheless, disease severity is not always related to the absence of protein expression, and most individuals that carry missense mutations express the mutated protein at levels ranging from low to normal. This, together with the fact that
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disease manifestations may differ considerably between patients with the same mutation, suggests that severity can be modulated by modifying genes affecting Treg function. As always when comparing the immune system between individuals, it should be kept in mind that phenotypic variability may also result from differences in the HLA haplotype and the lymphocyte repertoires [11]. Another factor that increases the complexity of the genotype/clinical phenotype relationship in IPEX is that other genetic defects affecting Treg function can originate an IPEX-like phenotype. These include loss-of-function mutations in CD25, STAT5B, and ITCH and gain-offunction mutations in STAT1 [12]. Furthermore, despite the progress attained in identifying the molecular basis of IPEX-like diseases, the underlying defects in a large percentage of individuals affected with these conditions remain obscure. Here, we present a study in which IPEX was suspected in three siblings, based on the clinical history of the family, starting 20 years ago. Identification of a novel mutation in the Phe367 residue of FOXP3 in the family led us to study the molecular impact of this and other mutations in the same residue on the structure of the FOXP3 protein. Functionally, this protein acts as a component of a dynamic multisubunit complex involved in histone modification and chromatin remodelling after T-cell receptor stimulation [13,14]. Its three-dimensional structure, in complex with NFAT1 (the DNA-binding domain of the nuclear factor of activated T cells-1) and a DNA fragment from interleukin-2 promoter, has been recently resolved experimentally [15]. Based on this structure, we analysed the spatial neighbourhood of Phe367 and its relationship with other FOXP3 mutations. Our results show that Phe367 is part of a rich network of hydrophobic interactions that are crucial for stabilizing both monomer and dimer structures. Many known IPEX-causing mutations are clustered in this region, suggesting a high sensitivity to sequence variations. The structural information provided here may be of value for future characterization of new mutations in this FOXP3 region. 2. Material and methods 2.1. Study sample The family reported here attended the Paediatric Infectious Diseases and Immunodeficiencies Unit of Hospital Universitari Vall d'Hebron (Barcelona, Spain). Informed consent was obtained from the family members studied, following the procedure of the Ethics Committee of Hospital Universitari Vall d'Hebron.
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3. Results 3.1. Family with suspected IPEX We report a non-consanguineous Spanish family with five siblings (three boys and two girls) from two different fathers (Fig. 1A). The family history included a maternal uncle who died due to hepatitis and diarrhoea at the age of three months. The three boys experienced earlyonset diarrhoea (clinical data in Table 1). The first boy, born in 1970, died at the age of two months because of probable infectious meningoencephalitis. He showed thymic hypoplasia, thrombocytopenia, pulmonary haemorrhage, and eosinophilic infiltrate in the gastrointestinal tract and adrenal glands. The second boy, born of a second marriage in 1986, had growth retardation, persistent diarrhoea, severe skin lesions and leucocytosis with eosinophilia in the first months of life. The infant died at the age of 13 months due to Klebsiella spp. sepsis. The third boy, born in 1988, showed growth retardation, hepatosplenomegaly, diarrhoea and severe eczema, leucocytosis with eosinophilia and had high IgE levels (N2000 kU/L). Several sepsis episodes caused by bacteria and fungi led to the child's death at the age of 13 months. Available hematologic and immunologic data of the patients are shown in Table 2. 3.2. Identification of the FOXP3 p.Phe367Val mutation Since the three affected siblings died more than 20 years ago, we were only able to trace DNA from the post mortem study of case 2. Unfortunately, the DNA was too degraded to successfully amplify all FOXP3 exons; hence, we resorted to study of the mother. Complete sequencing of the 12 exons of FOXP3 in the mother revealed a heterozygous nucleotide change (T N G) in exon 11, affecting the first position of codon 367 and leading to an amino acid change (phenylalanine to valine). This mutation had not been reported in the literature or databases, and, following the recommendations of the Human Genome Variation Society (HGVS), we named it c.1099 T N G/p.Phe367Val (Fig. 1B). To confirm that the mutation was present in at least one of the three affected siblings, we designed a specific pair of primers to amplify a short fragment of DNA containing the mutation from the sibling who had undergone post-mortem study. We successfully amplified and sequenced the FOXP3 fragment containing the mutation and confirmed that the boy had been a hemizygous carrier of the p.Phe367Val mutation. On study of the other family members, we found that the older sister was also a carrier, and genetic counselling was indicated (Fig. 1A). 3.3. Molecular impact of the F367V mutation
2.2. Mutation analysis Genomic DNA was extracted from EDTA-containing whole blood samples using a QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Polymerase chain reaction (PCR) to amplify the 12 exons of FOXP3 and their flanking regions was carried out using the primers and PCR conditions specified in Supplementary Table 1. Purified PCR products were sequenced on an ABI 3100 DNA Sequencer (Applied Biosystems, Foster, VA, USA). 2.3. Structure analyses and modelling All the structure analyses performed, such as the location of known mutations and contact computations, were done using the experimentally resolved structure of FOXP3 as reference (PDB code: 3QRF) [15]. To identify intradomain and interdomain residue-residue interactions, two residues were considered to be in contact when at least two of their atoms were located at a distance of 5 Å or less. Visual inspection to assess the impact of the mutations and creation of the structure images in the figures was done with the PyMol software package (PyMOL Molecular Graphics System, Version 1.7.4 Schrödinger, LLC).
To understand the molecular repercussions of the F367V mutation on FOXP3 function, we analysed its impact on the structure and predicted interactions of the protein. We found that the mutated residue (F367) locates within the experimentally determined structure of the DNA-binding domain (forkhead) of FOXP3, available in complex with NFAT1, the DNA-binding domain of nuclear factor of activated T cells1, and a DNA fragment from the promoter of interleukin 2 [15]. We analysed this structural information to provide a mechanistic view of the pathogenic effect of mutations at position Phe367. Subsequently, we sought additional support for this view by mapping the known FOXP3 pathological mutations to the forkhead structure [8]. 3.3.1. F367 is a key structural residue of the DNA-binding domain of FOXP3 The mutation described in this study (F367V) is not the only one occurring at position 367; two others, F367L and F367C, have been described. F367L has been reported in two separate IPEX patients: a Japanese infant with neonatal diabetes mellitus, intractable diarrhoea, liver dysfunction, thrombocytopenia, and sepsis who died at 16 weeks of age [16], and a French child with similar clinical manifestations [17] (Table 1). The group that performed the second study later reported the F367C mutation in a patient with severe protracted diarrhoea and
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Fig. 1. A. Main clinical characteristics of the three patients with IPEX and the family pedigree. B. Genetic analysis of the mother (carrier) showing the FOXP3 mutation c.1099TNG (p. Phe367Val) in heterozygous state.
protein-losing enteropathy [18] (Table 1). The description of additional mutations at F367 reinforced the idea that F367V would be pathogenic, and that position 367 is structurally and functionally critical. To test this idea, we analysed the structural environment of the wild-type residue, Phe367, using the forkhead structure in the NFAT1:FOXP3:DNA complex [15] (Fig. 2A). We found that Phe367 has an important role in both monomeric and dimeric structures (Fig. 2, B and C). Located in the H2 helix, Phe367 has 10 intra-monomer contacts of a mainly hydrophobic nature (Fig. 2B) that are an important part of the interaction network, stabilizing helices H1 and H2 in their relative orientation (Fig. 2, B and C). The significance of this contribution to the stability of the
forkhead domain is underlined by the relatively small size of the domain (87 amino acids). In accordance with this analysis, the FoldX stability computations, which provide an estimate of the change in protein stability occurring with a mutation [19], indicated that the three mutations at position 367 have a destabilizing effect, with values of 3.1 Kcal/mol (F367V), 1.7 Kcal/mol (F367L), and 4.7 Kcal/mol (F367C). In parallel, as we know that the functional form of FOXP3 is a dimer [20], we checked the location of Phe367 relative to the monomermonomer interface. We found that Phe367 contributes four hydrophobic residue-residue contacts (Fig. 2, C and D) to the forkhead functional dimer interface. This supports the idea that Phe367 has a functionally
Table 1 Clinical features of patients with mutations affecting the F367 residue of FOXP3. Reported onset F367V-P1 1 month
Endocrinopathy Enteropathy Skin Haematological manifestations anomalies
Recurrent infections
HSCT Outcome
Ref.
No
NA
No
This study
No
No
F367V-P2 1 month
No
Yes
Yes
F367V-P3 1 month
No
Yes
Yes
F367C
15
T1D
Yes
F367L
months 8 days
T1D
F367L
b6
T1D
NA
Yes. Bacteria (GNB)
No
Yes. Bacteria (Staphylococcus aureus, Enterobacter cloacae, E. coli, Acinetobacter). Fungi NA
No
No
Yes (eosinophilia) Yes (eosinophilia) NA
NA
Yes
Yes
Yes
Yes
No
Yes
Yes
No
Yes
NA
months GNB, gram-negative bacteria; HSCT, haematopoietic stem cell transplantation.
Died at 3 months Died at 13 months Died at 13 months NA Died at 4 months NA
This study This study [18] [16] [17]
R. Colobran et al. / Clinical Immunology 163 (2016) 60–65 Table 2 Hematologic and immunologic data of the IPEX patients.
Age Red blood cells (RBCs) Haemoglobin (gr/dl) Haematocrit (%) MCV (fL/red cell) Leukocytes (abs) Lymphocytes (%) Lymphocytes (abs) Monocytes (%) Monocytes (abs) Eosinophils (%) Eosinophils (abs) IgA (mg/dL) IgG (mg/dL) IgM (mg/dL) IgE (kU/L) Ratio CD4/CD8
Case 1
Case 2
Case 3
Reference values
– NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA
5 months 4.75 4 35.1 74 15,900 51 8109 4 636 10 1590 NA NA NA NA NA
11 months NA NA NA NA 35,000 28 9800 1 350 15 5250 18 407 82 N2000 2.1
3.8–4.8 10.5–13.5 33–39 77–95 5000–13,000 20–50 2300–5400 2–11 100–1000 0–5 50–250 11–153 196–1045 40–140 0–90 1.6–3.9
Abnormal values are shown in bold. MCV, mean corpuscular volume; Abs, absolute number; NA, not available.
relevant role, resulting from its contribution to the dimeric state of FOXP3, and that mutations at this location may have a disruptive effect on the dimer structure. The three mutations affecting the Phe367 residue result in a reduction in volume (F367V, −30 Å3; F367L, −11 Å3; F367C, −49 Å3), and it has been shown that interfacial cavity-creating mutations contribute to destabilize protein complexes through a combination of mechanisms, such as a hydrophobic effect or loss of van der Waals contacts [21]. In summary, structure analysis showed that Phe367 is involved in a rich interaction network related to stabilization of monomer and dimer structures. The relevance of this contribution is reinforced by the high conservation of Phe367 and its spatial neighbours on multiple sequence alignment of the FOXP3 family (Fig. 3). This is consistent with a
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pathogenic nature of the mutations under discussion, which may result from destabilization of the protein structure or direct disruption of the functional site [22,23]. 3.3.2. FOXP3 dimer interface is sensitive to sequence changes To provide further support for the results of the previous section, we mapped 29 known FOXP3 mutations [8] to the structure of the forkhead domain (Fig. 4; Supplementary Table 2). Many of them were located at the helices forming the dimer's interface (Supplementary Table 2) and involved a core of 8 aromatic residues known to stabilize the dimer [15]. Pathogenic mutations are listed for four residues of this core, and for at least one sequence neighbour of the remaining four residues (Supplementary Table 2). In addition, some of the mutated residues were within contact distance of Phe367 (Supplementary Table 2). This further supports the idea that Phe367 is part of a delicate interaction network whose integrity is fundamental for FOXP3 regulatory activity. 4. Discussion In this study, we describe the case of a family in which three members had a clinical phenotype consistent with IPEX syndrome. Sequencing of the FOXP3 gene showed a novel amino acid change (F367V) that had not been reported previously as a single-nucleotide polymorphism (SNP) or mutation. Prediction of the pathogenicity of the F367V mutation was important to establish an accurate molecular diagnosis and proper genetic counselling, as an increasing number of patients show a phenotype consistent with IPEX but do not harbour FOXP3 mutations [12]. Two other mutations affecting the same residue, F367L and F367C, have been described [16–18]. This makes F367 unique, because there are no other residues in FOXP3 where 3 different mutations have been reported in unrelated patients. This novel finding led us to analyse the impact of the mutation using an approach combining different tools. First, we studied the structural
Fig. 2. The structural environment of F367. A. Overall view of the location of Phe367 (shown using red spheres) relative to the FOXP3 forkhead (dimer, with monomers in lilac and light orange)-NFAT1 (green)-DNA (orange) complex. Relevant helices in the forkhead domains, (H1, H2 and H3) are indicated. B. Intramonomer and (C.) intermonomer contacts of residue Phe367 (shown in red). D. Detailed contact list for residue F367. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 3. Sequence conservation in the FOXP3 family. A. Local view of the multiple sequence alignment of FOXP3 in the neighbourhood of Phe367 (red arrow). Black arrows indicate the spatial residues of Phe367. B. Whole FOXP3 plot. Conservation is represented using Shannon entropy, a parameter routinely used for scoring pathogenic mutations, whose values range from 0 (complete conservation) to 4.32 (all amino acids are equiprobable at that position). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
neighbourhood of Phe367 and its relationship with known FOXP3 mutations, using the 3D FOXP3 reference structure [15]. The analysis showed that Phe367 is in a sensitive location, corresponding to the dimer interface of the FOXP3 functional form. This interface is densely affected with known pathogenic mutations [8], indicating its functional importance and suggesting a pathogenic role for F367V. To further support this conclusion, we tested the putative pathogenicity of F367V with two widely used prediction software packages, SIFT and PolyPhen-2. SIFT gave a deleterious prediction, in accordance with our structure analysis. The same happened with PolyPhen-2, once its multiple sequence alignment was corrected for the presence of a problematic sequence (see Supplementary Fig. 1). In summary, this extensive accumulation of evidence clearly indicates that the F367V mutation is pathogenic. In addition, the structure
analyses provide a relatively straightforward interpretation of the impact of the mutation, considering the functional value of the forkhead domain of FOXP3. This domain, which is the locus of Phe367, mediates DNA binding, acting as an activator/repressor [24]. FOXP3 binding requires a multimerization step, and the forkhead domain seems necessary for homo-multimerization of FOXP3 [15,25]. Previous studies have provided evidence that mutations in the forkhead domain of IPEX patients affect DNA binding [26,27]. Our work refines this view, indicating that mutations near or at the forkhead-forkhead interface may disrupt the network of interactions, impeding or hampering formation of the FOXP3-DNA complex and subsequently originating IPEX disease. This family case study illustrates the importance of reviewing clinical data in family members in whom a primary immunodeficiency is suspected. The in silico studies provided clues to evaluate the potential pathogenic effect of a genetic mutation in the absence of biologic material to study Tregs. Further descriptions of the molecular bases of primary immunodeficiencies will enable accurate preconception counselling in the relatives of children with these conditions.
Conflict of interest statement The authors declare no commercial or financial conflict of interest.
Acknowledgements
Fig. 4. Location of known FOXP3 mutated residues in the forkhead dimer. One monomer is shown in red, the other in light orange. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
The authors are thankful to the family members studied and the physicians involved in their care. We especially thank Pedro Domínguez Sampedro and Beatriz Valle del Barrio for their help in obtaining clinical data of the patients. We also acknowledge Celine Cavallo for their English language assistance. This study was funded by the Instituto de Salud Carlos III, grants PI11/1086 and PI14/00405, cofinanced by the European Regional Development Fund (ERDF), and by the Spanish Ministry of Economy and Competitiveness, grant BIO2012-40133.
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