Identification of the first patient with a confirmed mutation of the JAK-STAT system
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- Rosenfeld, R.G., Kofoed, E., Buckway, C. et al. Pediatr Nephrol (2005) 20: 303. doi:10.1007/s00467-004-1678-7
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Growth hormone insensitivity (GHI) has been attributable, classically, to mutations in the gene for the GH receptor. After binding to the GH receptor, GH initiates signal transduction through a number of pathways, including the JAK-STAT pathway. We describe the first patient reported with a mutation in the gene for STAT5b, a protein critical for the transcriptional regulation of insulin-like growth factor-I.
The syndrome of growth hormone insensitivity (GHI) was first identified by Laron and colleagues  almost 40 years ago. With the cloning of the cDNA for the GH receptor (GHR) twenty years later, it was demonstrated that classical cases of GHI were the result of mutations or complex deletions of the gene for the GHR . Because identification of cases of GHI at that time was dependent upon the demonstration of decreased serum concentrations of GH binding protein (GHBP), the extracellular, circulating domain of the GHR, the first identified mutations of GHR involved the extracellular domain [3, 4]. It subsequently became apparent, however, that a cohort of patients could be identified with an indistinguishable phenotype, including the failure to generate insulin-like growth factor-I (IGF-I) following administration of exogenous GH, but with normal serum concentrations of GHBP [5, 6]. Analysis of the GHR gene in such patients identified mutations affecting the transmembrane or intracellular domains of the receptor, or affected the ability of the GHR to dimerize, a critical step in GH action. Such patients, typically, have normal, or even increased, serum concentrations of GHBP, but the molecular defects of the GHR preclude normal GH signaling. Nevertheless, growth failure often was as pronounced, and serum IGF-I concentrations were as low, as in patients with classical GHI.
Recent years have seen increasing attention paid to such GHBP positive patients (patients with phenotypes consistent with classical GHI, but with normal serum levels of GHBP). Exclusion of patients with documented defects of the transmembrane or intracellular domains of the GHR still leaves a group of patients with clinical and biochemical evidence of GHI; such patients are clear candidates for post-GHR defects, involving the GH signaling cascade.
Several cases of short stature, with apparent GH resistance in the face of normal serum concentrations of GHBP have been evaluated for potential defects of the GHR signaling cascade. In at least two patients, studies in cultured cells appeared to suggest defective GH-induced tyrosine phosphorylation of STAT proteins, although no specific molecular defect was identified in either case [9, 10]. Ambrosio et al.  studied the STAT5b gene in 14 children with idiopathic short stature, but sequences proved to be normal in all cases. On the other hand, investigations involving targeted disruption of various STAT genes have indicated a significant role for STAT5b in mediating the growth-promoting action of GH in rodents . Of particular interest, only male mice were affected: males with STAT5b knockouts experienced a 30% reduction in size, while female mice were unaffected. Serum IGF-I concentrations were, similarly, reduced by approximately 30% in STAT5b-/- mice, compared to STAT5b+/+ mice, so that male mice with inactivated STAT5b had IGF-I levels comparable to wild-type females. As a result, wild-type females, and both males and females with STAT5b knockouts were equivalent in size, and all 30% smaller than wild-type males. Similar observations were made by Teglund et al. , who also observed that simultaneous knockouts of both the STAT5a and STAT5b genes affected growth in both male and female mice. These studies strongly supported a critical role for STAT5b in mediating sexually dimorphic GH-induced gene expression and growth in rodents.
First patient with a confirmed mutation in the gene for STAT5b
The opportunity to test the role of STAT5b in human growth occurred when a 16 year old girl from Argentina presented with severe postnatal growth failure (height −7.5 SD), combined with a history of hemorrhagic varicella, pneumocystis carinii, and lymphocytic interstitial pneumonia . The combination of severe growth retardation and immunodeficiency suggested the possibility of a signaling defect impacting the actions of both GH and cytokines. The fact that the parents were first cousins further supported a genetically transmitted disorder; of note is that classical GHI is an autosomal recessive disorder. Biochemical studies confirmed that the patient was GH sufficient, and severely IGF deficient [serum IGF-I, 36 ng/ml (nl 224–744 ng/ml); IGFBP-3, 874 ng/ml (nl 2500–4800 ng/ml); ALS, 2.9 ug/ml (nl 5.6–16 ug/ml)], with all three proteins poorly responsive to administration of exogenous GH. Serum concentrations of GHBP were normal, and sequencing of genomic DNA from the patient’s fibroblasts revealed no mutations of the GHR gene. While immunoblots for total STAT5 in patient fibroblasts indicated apparently normal levels, blots employing an antibody specific for the C-terminal domain of STAT5b showed no apparent protein and no phosphorylated protein. These results could be interpreted as an absence of STAT5b or a failure of the specific antibody employed to recognize an abnormal STAT5b epitope. RT-PCR and sequencing of the 2.4 kb coding region of STAT5b revealed that the patient was homozygous for a missense mutation in codon 630, resulting in the substitution of a proline (cct) for the wild-type alanine (gct). As anticipated, the parents proved to be heterozygous for the point mutation. Position 630 is in the src homology domain (SH2) of STAT5b, close to Y699, the tyrosine that is normally phosphorylated after the docking of STAT5b to the GHR. The A630P mutation introduces a novel Sty1 restriction site into the gene, permitting ready identification of the heterozygous and homozygous states.
The stability of the mutant STAT5b was evaluated by expressing the protein in a COS-7-cell expression system, stimulated by interferon-γ, a potent stimulus of both STAT5a and STAT5b phosphorylation . These studies demonstrated that the mutant A630P STAT5b could be stably expressed under such circumstances, but still could not be phosphorylated in response to either GH or interferon-γ. It is presumed that the mutation disturbs the functionality of the SH2 domain, potentially affecting phosphorylation and/or dimerization of STAT5b, and disrupting normal transcriptional regulation of IGF-I (see Fig. 1).
Interestingly, the identification of this novel mutation of the STAT5b gene results in the characterization of two new disease states: 1) GHI and primary IGFD ensuing from disruption of the GH signaling cascade; and 2) a new primary immunodeficiency disorder, presumably representing defective signaling of critical cytokine receptors. Of note is the fact that this female patient exhibited both severe growth retardation and IGF deficiency, thereby differentiating the human situation from the gender dimorphism characteristic of STAT5b and growth in rodents [8, 12]. It is to be anticipated that evaluation of related cases, such as growth failure and IGFD in the face of normal GH secretion, or the combination of immunodeficiency and short stature, will lead to the unmasking of additional defects of the GH signaling cascade [15, 16].