Novel Dominant-Negative GH Receptor Mutations Expands the Spectrum of GHI and IGF-I Deficiency

Abstract

Context: Autosomal-recessive mutations in the growth hormone receptor (GHR) are the most common causes for primary growth hormone insensitivity (GHI) syndrome with classical GHI phenotypically characterized by severe short stature and marked insulin-like growth factor (IGF)-I deficiency. We report three families with dominant-negative heterozygous mutations in the intracellular domain of the GHR causing a nonclassical GHI phenotype.

Objective: To determine if the identified GHR heterozygous variants exert potential dominant-negative effects and are the cause for the GHI phenotype in our patients.

Results: All three mutations (c.964dupG, c.920_921insTCTCAAAGATTACA, and c.945+2T>C) are predicted to result in frameshift and early protein termination. In vitro functional analysis of variants c.964dupG and c.920_921insTCTCAAAGATTACA (c.920_921ins14) suggests that these variants are expressed as truncated proteins and, when coexpressed with wild-type GHR, mimicking the heterozygous state in our patients, exert dominant-negative effects. Additionally, we provide evidence that a combination therapy of recombinant human growth hormone (rhGH) and rhIGF-I improved linear growth to within normal range for one of our previously reported patients with a characterized, dominant-negative GHR (c.899dupC) mutation.

Conclusion: Dominant-negative GHR mutations are causal of the mild GHI with substantial growth failure observed in our patients. Heterozygous defects in the intracellular domain of GHR should, therefore, be considered in cases of idiopathic short stature and IGF-I deficiency. Combination therapy of rhGH and rhIGF-I improved growth in one of our patients.

Keywords: GH/IGF-I therapy; dominant-negative GHR mutations.

Figure 1.

Heterozygous GHR variant segregation in affected families. (a) P1, c.964dupG; (b) P2, c.920_921ins14; (c) P3, c.945+2T>C; and (d) P4, de novo c.899dupC [15]. Arrow, proband.

Figure 2.

The growth charts of the patients carrying heterozygous GHR variants. (a) P1, c.964dupG; (b) P2, c.920_921ins14; (c) P3, c.945+2T>C; and (d) P4, c.899dupC [15]. The time frame of rhIGF-I, rhGH, and rhIGF-I + IGFBP3 treatments are indicated on the growth curves. CDC, Centers for Disease Control and Prevention.

Figure 3.

Heterozygous GHR variants identified in affected patients. (a) Schematic representation of the hGHR protein showing the various domains and the location of the heterozygous mutations, c.899dupC, c.920_921ins14, c.945+2T>C, and c.964dupG. TM, transmembrane. (b) Electropherograms of the WT GHR and P1 carrying the c.964dupG variant. The reverse primer was used for sequencing and the electropherogram presented is the complement, forward sequences. Heterozygosity is, therefore, visualized 5′ to the highlighted G nucleotide. (c) DNA sequence (5′ to 3′) and corresponding amino acids showing the duplication of the nucleotide G (underlined) in the c.964dupG variant and the resulting frameshift and early termination (stop). (d) Electropherograms of the forward WT GHR and P2 showing the c.920_921ins14 variant. (e) DNA sequence (5′ to 3′) and corresponding amino acid sequence showing the insertion of 14 bp at nucleotide 921 of GHR cDNA (boxed) in the c.920_921ins14 variant, the resulting frameshift, and stop. (f) Electropherograms of the exon 9–intron 9 junction from WT GHR and P3 carrying the c.945+2T>C variant (arrow); the mother, but not the father, carried the variant, as did the maternal grandfather (data not shown). (g) Schematic, c.945+2T>C splicing out of exon 9. *Position of the mutation. The cDNA sequence and translated products of WT (GHR) and the c.945+2T>C variant are shown (bottom) along with the splice junctions. (h) DNA gel of GHR mRNA RT-PCR products from a normal control and P3, amplified using primers from exon 8 to 10. The expected product size is 187 bp (control). The presence of a smaller 117-bp band in the proband confirms deletion of exon 9. (i) A larger sample (20 µL) of the RT-PCR product better demonstrates the presence of two products (187 bp and 117 bp) carried by P3.

Figure 4.

GH-induced STAT5B activities in HEK293 cells transiently transfected with the WT and mutated GHR variants regenerated by site-directed mutagenesis. Input hGHR constructs are indicated. The fully glycosylated GHR, truncated GHR, PAK STAT5 (pSTAT5), and total STAT5B are indicated by arrows. (a) Immunoblot analysis of GHR, STAT5B, and GH-induced STAT5B activation in cells transfected with the c.964dupG variant. (b) Immunoblot analysis of GHR, STAT5B, and GH-induced STAT5B activation in cells transfected with the c.920_921ins14 variant. (c) GH-induced transcriptional activities as measured by relative pGHRE-LUC activities. The data are mean ± standard error of the mean of at least three experiments. The results of mutant only (c.964dupG or c.920_921ins14) or WT + mutant cotransfections were compared with WT. *P < 0.01, **P < 0.001.