PMC8072467_01
GHR gene variants in patients with Laron syndrome. Patient 2 (A) carried the c.808A>G (p.I270V) homozygous mutation. Patient 3 (B) harbored the c.766C>T (p.Q256*) heterozygous mutation. Patient 4 (C, D) had GHR gene compound heterozygous mutations: c.587A>C (p.Y196S) and c.1707_1710del (p.E570Afs*30). Pedigree analysis of patient 4 (E) revealed that the c.587A>C variant was inherited from his mother, while the c.1707_1710del variant was inherited from his father.
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Table 3  and  Figure 1 . Two single nucleotide polymorphism (SNP) sites, c.1483C>A (p.P495T, rs6183) and c.1630A>C (p.I544L, rs6180), were detected in Patient 1. Patient 2 carried a novel homozygous mutation, c.808A>G (p.I270V) as shown in  Figure 1A , and neither of his parents had the c.808A>G mutation. A novel heterozygous nonsense mutation, c.766C>T (p.Q256*) was identified in patient 3 as shown in  Figure 1B , and the C>T base substitution led to a premature stop codon, which produced a truncated GHR protein. Patient 4 carried GHR gene compound heterozygous mutations including a novel heterozygous mutation, c.1707_1710del (p.E570Afs*30) in exon 10 ( Figure 1D ), which was inherited from his father and a previously reported heterozygous mutation, c.587A>C (p.Y196S, rs747888560) in exon 6 ( Figure 1C ), which was inherited from his mother as presented in  Figure 1E .
Immunofluorescence images of HepG2 cells transfected with GHR wild type (WT) and mutant expression plasmids (Q256*, E570Afs*30 and Y196S). The GHR protein was labeled by anti-HA primary antibody in green, the nucleus was labeled by DAPI in blue, and the merged images were displayed in the right column.
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Immunofluorescence images of HEK293T cells transfected with GHR wild type (WT) and mutant expression plasmids (Q256*, E570Afs*30 and Y196S). The GHR protein was labeled by anti-HA primary antibody in green, the nucleus was labeled by DAPI in blue, and the merged images were displayed in the right column.
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GHRs were again conducted in HEK293T cells by the same transient transfection method and immunofluorescence assays as shown in  Figure 3 , and the localization of the GHR-WT and mutant GHRs were similar to that of HepG2 cells. GHR-WT proteins in HEK293T cells presented with a uniform distribution on cell surface as observed in HepG2 cells transfected with GHR-WT.
(A, B) Expression of GHR protein in HepG2 cells transfected with pcDNA3.1(+) empty vector, GHR wild type (WT) and mutant expression plasmids (Y196S, Q256* and E570Afs*30). The GHR protein has a molecular weight of around 130 kDa. Protein bands corresponding to 130 kDa could be detected in HepG2 cells transfected with Q256* and E570Afs*30, and the relative expression of both of them were significantly decreased compared to GHR-WT (C, P<0.05). HepG2 cells transfected with Q256* mutant plasmid produced a truncated protein, with a molecular with of around 43kD. However, no truncated protein was detected in HepG2 cells transfected with E570Afs*30 mutant plasmid.
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GHRs could be detected in HepG2 cells transfected with pcDNA3.1(+) empty vector, and the molecular weight of GHRs were about 130 kDa. GHR-WT proteins were overexpressed compared to cells transfected with empty vector and the difference was statistically significant ( Figure 4C , P<0.05). The GHR protein levels in HepG2 cells transfected with GHR-Y196S were significantly lower than that of GHR-WT and decreased by 19.65% as shown in  Figure 4C  (P<0.05). Specifically, unlike GHR-Y196S, HepG2 cells transfected with GHR-Q256* mutant expression plasmid produced a truncated protein, with a molecular weight of approximately 43 kDa as presented in  Figure 4B . HepG2 cells transfected with GHR-E570Afs*30 mutant plasmid also had significantly diminished GHR protein expression at a molecular weight of 130kDa, which was decreased by 60.22% when compared to GHR-WT as presented in  Figure 4C  (P<0.05). However, no visible truncated GHR protein band was identified in western blot gel.
Phosphorylated STAT5 (p-STAT5) expression in HepG2 cells transfected with wild type GHR (WT), mutant GHR Y196S, Q256* and E570Afs*30 after 100ng/ml rhGH was added in the cell culture medium for 30 minutes. Both p-STAT5 and total STAT5 (t-STAT5) had a molecular weight of around 95 kDa (A, B). The ratio of p-STAT5 to t-STAT5 was converted to gray-scale value by Image J. The expression of p-STAT5 was significantly decreased in GHR-Y196S, GHR-Q256* and GHR-E570Afs*30 when compared to GHR-WT as shown in (C) (P<0.05).
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As shown in  Figures 5A, B , both total STAT5 (t-STAT5) and p-STAT5 were detected at a molecular weight of 95 kDa in HepG2 cells. As shown in  Figure 5C , the levels of p-STAT5 proteins were significantly decreased in HepG2 cells transfected with GHR-Y196S mutant plasmid in comparison with cells transfected with GHR-WT, and decreased by 32.67% (P<0.05). In consistent with the results of GHR-Y196S, the levels of p-STAT5 proteins were also significantly reduced in HepG2 cells transfected with GHR-Q256* and GHR-E570Afs*30 mutant plasmids and reduced by 40.57% and 29.63%, respectively, as shown in  Figure 5C , which indicated an impaired post-receptor signal transduction.