Characterization of Immunodeficiency in a Patient With Growth Hormone Insensitivity Secondary to a Novel STAT5b Gene Mutation
STAT5 proteins are components of the common growth hormone and interleukin 2 family of cytokines' signaling pathway. Mutations in the STAT5b gene, described in 2 patients, lead to growth hormone insensitivity that resembles Laron syndrome. Clinical immunodeficiency was also present, although immunologic defects have not been well characterized thus far. Here we describe a 16-year-old girl who suffered generalized eczema and recurrent infections of the skin and respiratory tract since birth. She also suffered severe chronic lung disease and multiple episodes of herpetic keratitis. Clinical features of congenital growth hormone deficiency were observed, such as persistently low growth rate, severely delayed bone age, and postnatal growth failure resulting from growth hormone resistance. This combined phenotype of growth hormone insensitivity and immunodeficiency was attributable to a homozygous C→T transition that resulted in a nonsense mutation at codon 152 in exon 5 of the STAT5b gene. This novel mutation determined a complete absence of protein expression. The main immunologic findings were moderate T-cell lymphopenia (1274/mm3), normal CD4/CD8 ratio, and very low numbers of natural killer (18/mm3) and γδ T (5/mm3) cells. T cells presented a chronically hyperactivated phenotype. In vitro T-cell proliferation and interleukin 2 signaling were impaired. CD4+ and CD25+ regulatory T cells were significantly diminished, and they probably contributed to the signs of homeostatic mechanism deregulation found in this patient. This new case, in accordance with 2 previously reported cases, definitely demonstrates the significant role of the STAT5b protein in mediating growth hormone actions. Furthermore, the main immunologic findings bring about an explanation for the clinical immunodeficiency features and reveal for the first time the relevant role of STAT5b as a key protein for T-cell functions in humans.
Cytokines such as interleukin (IL)-2, IL-4, IL-7, IL-9, IL-15, IL-21, and growth hormone (GH) mediate their responses through activation of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway.1
Although the GH receptor (GHR) and the IL-2 receptor (IL-2R), in the presence of their specific ligands, recruit and activate different cytosolic JAK proteins (such as JAK2 for GHR and JAK1/3 for IL-2R), they share STAT5 proteins for their signaling pathways.1
The STAT5 proteins a and b show 96% homology2 and are encoded by 2 distinct but closely related genes, STAT5a and STAT5b. They have been described as having both redundant and nonredundant roles in the response of cells to different growth factors.3
The exclusive role of STAT5 in mediating GH actions has been demonstrated in mice3,4 and recently in humans5,6 in 2 nonrelated patients with STAT5b mutations. These subjects presented GH insensitivity, resembling Laron syndrome, and also associated with recurrent severe infection diseases. However, it is well known that Laron syndrome is not associated with immunologic defects.7
In the immune system, the importance of STAT5 proteins as mediators of the IL-2 family of cytokines has been established on the basis of different mouse models, supporting their role in development, homeostasis, and proliferation of different lymphocyte populations.8–11
A severe immunologic phenotype was found in double–knock-out STAT5a/b-deficient mice.11 Some of them exhibited autoimmune pathology,9 similar to mice lacking IL-2 or its receptor components: IL-2Rα or IL-2Rβ.12–14 Because the disease was associated with decreased numbers of CD4+CD25+ regulatory T cells (Tregs), which are required to suppress immune response and prevent autoimmunity,15 it has been proposed that STAT5 might be critical for the maintenance of tolerance in vivo.9 Particularly, the Stat5b-deficient mouse has less dramatic immunologic alterations than the double knock-out, characterized by decreased numbers of T cells, low proliferation of lymphocytes to mitogens, IL-2 or IL-15 activation, and very low natural killer (NK) cell number and function.16
We report here a novel STAT5b gene mutation that totally precluded protein expression in a patient with GH insensitivity and immunodeficiency. A thorough characterization of the immunodeficiency showed moderate lymphopenia, with very low numbers of NK and γδ T cells (which contribute to mucosal immunity and host defense against intracellular pathogens), as well as functional defects of T cells. The finding of a reduced number of Tregs would suggest that in humans, as in mice,17,18 Tregs require the activation of STAT5.
The patient, a 16-year-old girl, was the first of 2 children of nonconsanguineous parents. She was born with a birth weight of 2500 g. She has had severe growth failure since the first year of life. She was first referred to another institution at the age of 6 years, at which time her height was 83.6 cm (SD score; [SDS]: −6.17), her weight was 9800 g (relative weight: 89%), and her bone age was 2.5 years (Fig 1).
Since her first months of life she presented generalized eczema and recurrent severe infections of the skin and respiratory tract that required multiple hospitalizations. She also presented recurrent episodes of pneumonia, for which she was hospitalized and treated with intravenous antibiotics. No microbiologic isolations were reported.
She had been suffering from chronic diarrhea since she was 2 months old. Gastroenterological and laboratory evaluations ruled out cystic fibrosis and celiac disease. Ascaris lumbricoides and Enterobius vermicularis were found at different times; to our knowledge, no other organisms were isolated. At the age of 4 years she had prolonged varicella complicated with bacterial infection. Since the age of 10 years, she has had recurrent herpes zoster keratitis and uveitis of the left eye, with progressive loss of visual acuity.
No family history of immunodeficiency or growth failure was reported. However, short stature was observed in her mother. Paternal and maternal heights were 160 cm (SDS: −2.2) and 142.3 cm (SDS: −3.3), respectively. Her 10-year-old brother had normal height. At 16 years of age she was hospitalized because of respiratory distress and was referred to our immunology and endocrinology units. Clinical features of congenital GH deficiency were observed: prominent forehead, saddle nose, and high-pitched voice, with no signs of pubertal onset. She had a persistently low growth rate and severe growth failure (height: 102.7 cm [SDS: −9.91]; weight: 12 kg [SDS: −6.7]), and her bone age was severely delayed (7 years old). Basal serum triiodothyronine, thyroxine, thyrotropin, cortisol, luteinizing hormone, and follicle-stimulating hormone were normal for age and pubertal development. However, here serum prolactin level was persistently high in 3 different samples drawn with an interval time of 1 month (168, 102.3, and 143 ng/mL).
She had evidence of chronic lung disease with signs of chronic hypoxemia (clubbing). Active herpes keratitis and severe eczema were also present. Pharyngeal swabs and blood and sputum cultures were negative for virus, fungus, bacteria, and mycobacteria.
Serology analysis showed negative results for HIV, cytomegalovirus, and Epstein-Barr virus (EBV) immunoglobulin (Ig)M but positive results for cytomegalovirus and EBV IgG. The thorax computed tomography scan showed a ground-glass appearance with generalized micronodular images and bronchiectasis in the left lower lobe. Pulmonary-function tests showed mixed, restrictive and obstructive ventilative insufficiency of moderate degree. She was treated empirically with antibiotics without any response.
Lymphoid interstitial pneumonia was suspected; a lung biopsy was indicated, but the parents refused the test. Since then, the patient's pulmonary function has worsened, and permanent oxygen has been required. Because of the long history of recurrent infections, chronic lung disease, and severe growth retardation, immunodeficiency and GH deficiency or insensitivity was suspected.
The patient was lost to follow-up. Treatment with high doses of prednisone was started in another institution and, after 3 months, a partial improvement of the respiratory condition was observed (E.M. Rivas, verbal communication, 2005).
Our study was approved by the institutional review board of the Garrahan Pediatric Hospital. Informed consent, as well as assent, were obtained from the parents and patient, respectively.
Serum Hormone Studies
Twenty-minute–interval basal serum GH was determined by a commercial chemiluminescent enzyme immunometric assay (Diagnostic Products Corporation, Los Angeles, CA).19
Insulin-like growth factor I (IGF-I) and IGF-binding protein 3 (IGFBP-3) levels were determined at basal conditions and during 5 consecutive days of treatment with 0.1 IU/kg per day of recombinant human GH. IGF-I was measured by radioimmunoassay after acid-ethanol extraction of serum,20 and IGFBP-3 was measured by immunoradiometric assay with a diagnostic kit (Diagnostic System Laboratory, Webster, TX).
Serum prolactin was determined by microparticle enzyme immunoassay using a diagnostic kit (Axsym System; Abbott, Abbott Park, IL).
Phenotypic analysis was performed after 3 color flow-cytometry (FC) standard methods21 using monoclonal antibodies (mAbs) from Becton Dickinson (San Jose, CA) and a FACSort instrument (Becton Dickinson) equipped with CellQuest software (Becton Dickinson).
Perforin (PharMingen, San Diego, CA) in NK cells was determined on peripheral blood mononuclear cells (PBMCs) preincubated 24 hours with either medium alone or plus recombinant IL-2 (rIL-2) (50 IU) by standard intracellular staining methods.21 Analysis was performed by gating the population-specific cell antigens, CD3−CD56+ and reporting the mean channel of fluorescence intensity (MCFI).
IL-2–Induced Phosphorylation of STAT5 Protein
On the basis of the method described by Fleisher et al,22 PBMCs were used for the evaluation of STAT5 phosphorylation in basal conditions or under stimulation with 1000 IU of rIL-2. Permeabilized cells were incubated with anti-STAT5 (BD Transduction Lab, Lexington, KY), antiphospho-STAT5 (Zymed Laboratories, San Francisco, CA), and isotype control antibody (MOPC 31C Sigma, St Louis, MO). Data were reported as the percentage of lymphocytes staining positive for STAT5 or phospho-STAT5 versus cells stained with isotype control. The data of MCFI were analyzed also.
Lymphocytes Functional Assay
Lymphocyte proliferation was assessed as described previously23 on PBMC culture using the [3H]thymidine-incorporation method with optimal concentrations of mitogens (phytohemagglutinin, anti-CD3, Staphylococcus enterotoxin B, phorbol myristate acetate, and ionomycine) for 3 days and specific antigens (purified protein derivative, Candida, and tetanus toxoid) for 6 days. To asses IL-2 proliferative response under mitogenic stimulation, rIL-2 (50 μg/mL, 50 IU) was added.
Cytotoxic NK activity was measured on a 51Cr-release assay as described previously24 after 24 hours of preincubation of PBMCs either with medium alone or under rIL-2 (50 IU) stimulation. The percentage of cytotoxicity was determined according to the following formula: % cytotoxicity = (mean patient cpm − mean spontaneous cpm)/(mean cpm of maximum − mean spontaneous cpm) × 100.
Western immunoblotting was conducted by using mouse mAbs anti-human-STAT5 (BD Transduction Lab), anti-human-STAT5b (Santa Cruz Biotechnology, Santa Cruz, CA), and anti-human-actin (Santa Cruz Biotechnology) as an internal control.
EBV immortalized B-cell lines derived from the patient and a normal control were solubilized in radioimmunoprecipitation assay lysis buffer. Total protein concentration was determined by using a commercial protein assay (Bio-Rad, Hercules, CA). Samples were electrophoresed on 7% sodium dodecyl sulfate/polyacrylamide gels and electroblotted onto nitrocellulose membranes (Hybond ECL, Amersham, Piscataway, NJ). Proteins were detected with ECL chemiluminescence reagents (Amersham).
DNA Isolation, Amplification, and Sequencing
Each coding exon of the STAT5b gene and flanking intronic regions were amplified from the patient and relative genomic DNA using primers reported by Kofoed et al.5 Each purified product (QiaQuick polymerase chain reaction purification kit, Qiagen, Valencia, CA) was used as a template for direct sequencing (automated ABI PRISM 310 capillary sequencer, Applied Biosystems, Foster City, CA). The nucleotide sequences obtained were compared with those for contig DNA containing the STAT5b gene (NT_010755 accession number gi51474257).
All patient data were compared with age-related reference values of our immunology laboratory at the Hospital de Pediatría Garrahan.
Differences between means were evaluated by using the Bonferroni test after 1-way analysis of variance.
Evaluation of the GH–IGF-I Axis
Basal GH was evaluated in 4 different serum samples at 0 (4.2 ng/mL), 20 (4.6 ng/mL), 40 (6.6 ng/mL), and 60 minutes (4.2 ng/mL). According to a previous report,19 6 ng/mL was the cutoff to define a normal serum GH response to pharmacological test. Thus, a basal GH value reaching 6.6 ng/mL was sufficient to discard the diagnosis of GH deficiency. In addition, the diagnosis of severe GH insensitivity was made, because no detectable IGF-I or IGFBP-3 was measured under basal conditions or after recombinant human GH treatment during the IGF-I-generation test.
Moderate lymphopenia and hypergammaglobulinemia, mainly of IgG and IgE isotypes, was observed (Table 1). Serologic evaluations showed positive IgG antibodies for varicella herpesvirus, hepatitis A virus, EBV, cytomegalovirus, and herpes simplex virus I and II. Serum antibodies specific to recall antigens (tetanus toxoid and Pneumococcus) were within the reference range (Table 1), showing integrity of the antibody function. Complement proteins were below the reference levels, and a high titer (1/1000) of antinuclear antibody was detected.
The patient had moderate T-cell lymphopenia, with numbers of CD4 and CD8 T cells in the lower limit and below the reference range, respectively, but within the reference CD4/CD8 ratio. B cells were within the reference range. It is notable that an extreme reduction in percentages and in absolute counts of NK and γδ T cells was found (Table 1).
Analysis of the CD4+CD25+ Treg population was based on the definition of Tregs by Baecher et al.25 Despite the fact that T cells were hyperactivated, a minor population with high expression of CD25 could be detected that represented only 1.16% ± 0.36% (n = 3) of the total CD4 T cells (Fig 2C). According to our age-matched reference values of 4.04% ± 0.26% (n = 7), they were clearly diminished (P < .01). These cells constitutively expressed cytotoxic T-lymphocyte–associated protein 4 (CTLA4) and were CD62L+high, indicating that, phenotypically, they were truly Tregs25 (data not shown).
In vitro T-cell proliferation is shown in Table 2. Proliferation to mitogens (phytohemagglutinin, S enterotoxin B, phorbol myristate acetate/ionomycine, and anti-CD3) was consistently low and could not be restored to reference values despite rIL-2 stimulation. After natural exposure or vaccination, absence of proliferative response to recall antigens (tetanus toxoid, purified protein derivative, and Candida) was observed (Table 2). These results support the concept of a general functional T-cell defect besides the defect in the IL-2 signaling pathway.
The presence of high T-cell activation, hypergammaglobulinemia, antinuclear antibodies, and diminution of CD4+CD25+ Tregs suggests a defect in the peripheral homeostasis mechanisms.
To analyze the IL-2 signaling pathway, STAT5 phosphorylation under IL-2 stimulation was evaluated by FC.
In the patient's lymphocytes, as shown in Fig 3A, STAT5 protein phosphorylation in response to high concentrations of rIL-2 was reduced to less than half of the control MCFI value (206 vs 462 ± 51.8 [n = 3]).
The expression of STAT5 proteins was reduced compared with the normal control (MCFI value: 291 vs 464 ± 62.7 [n = 3]) (Fig 3B).
As a whole, these results suggest that the functional impairment in IL-2-mediated signaling might be the consequence of an alteration in either of the 2 STAT5 proteins.
To evaluate the expression of both STAT5a and STAT5b proteins, immunoblot analysis was performed by using an anti-STAT5 mAb and 7% sodium dodecyl sulfate/polyacrylamide gels that discriminate STAT5a (91 kilodaltons) from STAT5b (84 kilodaltons) proteins, as shown in Fig 4A. The STAT5b protein is not detected at all in the patient's sample, whereas the related STAT5a protein level is comparable to those found in the normal B-cell line. The absence of STAT5b was confirmed with an immunoblot analysis using a specific anti-human STAT5b mAb (Fig 4B).
To determine the genetic basis for the apparent lack of immunodetectable STAT5b, STAT5b coding exons were sequenced. The results revealed a novel single-nucleotide change, a homozygous C→T transition, that resulted in a nonsense mutation at codon 152 (R152X). The parents and brother were heterozygous for this mutation. The stop-codon mutation in the STAT5b gene is in exon 5, which encodes the coiled-coil domain, very close to the NH2-terminal domain, predicting that the protein should not be immunodetectable.
NK Activity and Perforin Expression Induced by IL-2
Because IL-2 is a potent in vitro stimulator of NK cytolytic activity, we evaluated if this function was affected in the absence of the STAT5b protein. A clear 2.5-fold (from 8% to 20%) and 10-fold (from 3% to 30%) increase in cell lysis in the presence of IL-2,was found in the patient when compared with basal conditions. In the control samples, an average of a twofold increase in NK activity was observed (from 29 ± 17.2 to 60 ± 22.9 [n = 5]; P = .042). Although the range of lysis enhancement in the patient was higher, it did not reach the normal control level of cytotoxic activity, possibly influenced by the low NK cell number.
Because perforin is one of the key molecules for NK-cell–mediated cytolysis, the upregulation of the protein expression by IL-2 was also analyzed by FC on NK cells. Corresponding to the NK cytotoxicity results, IL-2 increased the protein MCFI value from 136 ± 14.8 to 179.5 ± 10.6 (n = 3; P = .014) (controls: 147 ± 18.0 to 174.7 ± 23.3 [n = 4]). These data suggest that STAT5b is not a limiting factor for mediating IL-2 actions in NK cells.
The studies described for this patient lend support, for the first time, to the relevance of STAT5b not only in the human GH signaling cascade but also in the cytokine-mediated immune response. We describe here a novel mutation in the STAT5b gene in a patient with a combined phenotype of GH insensitivity and immunodeficiency secondary to lymphocyte functional alterations.
The nonsense mutation, located in exon 5 of the STAT5b gene, predicts a severely truncated protein that retains only the NH2-terminal domain. Because it lacks the tyrosine-activation domain, it is expected to be devoid of any biological activity. Immunoblot analysis failed to detect the STAT5b protein, probably because the mutant STAT5b product suffers a rapid degradation or it is not expressed at all.
As in the 2 patients described previously,5,6 basal normal-to-high concentration of serum GH and extremely low serum IGF-I and IGFBP-3 levels were found in this case. Moreover, exogenous GH treatment did not further increase serum IGF-I or IGFBP-3 levels, confirming that STAT5b abnormality affects GHR signaling cascade. Similarly to the other 2 cases with STAT5b gene mutations5,6 and to those observed in GHR deficiency,7 near-normal size at birth and severe postnatal growth failure were observed. Therefore, this postnatal growth pattern reinforces the relevance of the GH-GHR-STAT5b-IGF-I pathway in human postnatal growth. In this regard, the STAT5b protein in STAT5 knock-out models is also essential for GH biological response in terms of postnatal growth and IGF-I expression. However, the postnatal growth observed in the patients is in contrast to that of mice, in which only male animals are affected.4
The persistently high hyperprolactinemia suggests a state of prolactin resistance. In this regard, prolactin specifically stimulates phospho-STAT5b in cultured hypothalamic dopaminergic neurons.26 In addition, a specific intracellular pathway of prolactin-negative feedback in the hypothalamus is mediated by STAT5b, as it has been demonstrated in STAT5b knock-out mice, showing grossly elevated serum prolactin concentrations.27 Therefore, the patient's hyperprolactinemia suggests that STAT5b plays a role in mediating the negative feedback action of prolactin also in humans.
The relevant role of STAT5 proteins in the immune system thus far has been defined only in mouse models. Similarly to the 2 patients reported, in our case clinical signs of immunodeficiency were clearly found, because she suffered from recurrent pulmonary infections, persistent herpesvirus infection, chronic eczema, and diarrhea. However, the immune system abnormalities in the 2 patients with STAT5b gene mutations already reported have not been well characterized.
In our patient, similar to the STAT5b knock-out16 mouse, phenotypic alterations, moderate T-cell lymphopenia, and highly reduced NK and γδ T cells were found. Moreover, the T-cell–hyperactivated status resembles the STAT5a/b double–knock-out mice11 and, interestingly, also the knock-out mice for IL-2Rβ, the signaling-IL-2R chain that serves as a STAT5 docking site.28
T cells were functionally defective, which was clearly documented by the in vitro low proliferative response to mitogens and antigens. Even in the presence of rIL-2, no proliferation improvement was observed. These results reflect a generalized defect in T-cell functions that could be a consequence of both defective STAT5 signals through T-cell receptor (TCR)29 and IL-2R. Moreover, resistance to IL-2 takes place even in the presence of normal amounts of STAT5a and one half of IL-2–STAT5 phosphorylation, which reinforces the concept that STAT5a and STAT5b are not completely redundant.3 Thus, different from mice,11 a full STAT5 function would be necessary in humans for a complete immune response.
On the other hand, we observed that the expression of IL-2Rα chain (CD25) could not be sustained in our patient's T cells in culture even in the presence of rIL-2 (data not shown). The fact that IL-2Rα is controlled by IL-2 through STAT5 proteins16,30 suggests that the expression of a high-affinity receptor is also affected, and it might have an influence in the lack of response to IL-2.
Nevertheless, the possible impact of the absence of STAT5b on impaired thymic function should also be considered in the context of T-cell lymphopenia, skewed CD45RA/CD45RO ratio, proliferative defects, and chronic infection. Additional studies designed to evaluate thymic output could clarify this point.
Contrary to T cells, B lymphocytes were normal in number and function, suggesting that STAT5b protein is mainly relevant in mediating T-cell–lineage functions. However, the presence of hypergammaglobulinemia and anti-nuclear antibodies, even without clinical autoimmune disease, suggests a deregulation of B cells, probably as a result of an impaired control by defective T cells.
Several evidences show that IL-2 is not only an important growth factor for T cells through TCR stimulation but also exhibits a critical role in the maintenance of in vivo tolerance to self-antigens, because spontaneous accumulation of activated T cells and autoimmunity develop in IL-231 or IL-2Rβ14 knock-out models or downstream signaling-component knock-out models such us STAT5a/b−/− mice.9,11 However, the role of STAT5 proteins in the regulation of the immune response in humans has not been clearly established. One of the mechanisms for maintenance of peripheral tolerance by IL-2 is through the generation of CD4+CD25+ Tregs.32 In this regard, it has been described in mice that STAT5 protein signaling is required for IL-2 Treg development and homeostasis.17,18 In knock-out mouse models for STAT5a/b, the reduction of this T-cell subset was considered to contribute to the loss of tolerance detected in a group of them. The reduction in Tregs correlated with a high rate of cell death in this population.9
Our findings are compatible with a deregulation of the mechanisms of tolerance in our patient, such as the detection of high titers of antinuclear antibody, hypergammaglobulinemia, and T-cell activation. However, hypergammaglobulinemia and T-cell activation could also be secondary to infections. Moreover, on the basis of the characteristics of the pulmonary disease of the previously reported patients,5,6 we suspected a similar chronic pulmonary illness as a consequence of lymphocytic infiltration. Because a significant decrease in Treg number that might contribute to this phenotype was found, we propose that in humans complete STAT5 function is required for establishing normal tolerance, although additional studies are necessary to confirm this proposal.
Similarly to STAT5 knock-out mice,11,16 a severe reduction in our patient's NK-cell number was found, suggesting that either development or homeostasis of these cells in the periphery depend on STAT5b. For both mechanisms, dependence on the IL-15/STAT5 pathway seems to be crucial.33 However, in contrast to the STAT5b knock-out model, the patient's NK cells showed a response to IL-2, increasing NK cytotoxic function, and upregulating perforin. Although this could be secondary to the activation of an alternative pathway,34 the recurrent herpetic infections in our patient suggest an inadequate NK surveillance, considering the crucial role of these cells in innate immune defense against viruses.35 Moreover, primary immunodeficiencies with selective deficiency in NK cells suffer mainly infections with herpes family viruses.36,37
We have demonstrated T-cell–function defects, a low number of NK and γδ T cells, IL-2 signaling defects at the T-cell level but preserved in NK, and deregulation of homeostasis (probably related to a decreased number of Tregs) in a patient with GH insensitivity resulting from a novel mutation in the STAT5b gene.
This mutation results in alterations in the signaling pathways of GHR, cytokine receptors related to T lymphocytes such as IL-2 receptor, and probably prolactin receptor. This is explained biologically by the fact that STAT5b is a signal transduction protein shared by all these factor receptors.
This work was supported by grants from Consejo Nacional e Investigaciones Científicas y Técnicas and Fondo para la Investigación Científica y Tecnológica of Argentina.
We thank Dr Ron Rosenfeld for valuable advice.
- Accepted June 13, 2006.
- Address correspondence to Alicia Belgorosky, MD, PhD, Hospital de Pediatría Garrahan, Endocrinology Service, Combate de los Pozos 1881, C1245AAM Buenos Aires, Argentina. E-mail:
The authors have indicated they have no financial relationships relevant to this article to disclose.
Drs Bernasconi and Marino contributed equally to this work.
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- Copyright © 2006 by the American Academy of Pediatrics