PMC8378174_01
PMC8378174_02
A novel mutation of MKRN3 in a central precocious puberty (CPP) patient. (A) Pedigree of a family with a novel c.227G>A mutation in the human MKRN3 gene. (B) Partial sequencing chromatographs of the MKRN3 gene of the CPP patient and her family members. Squares indicate male family members; circles, female family members; black, individuals with CPP; gray, individuals CPP status unknown; symbol with black dot inside, asymptomatic carriers; NM, no mutation allele. The arrow indicates the proband.
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F1
Genes panel analysis indicated that the proband had a novel MKRN3 gene mutation (Figure 1A). Her mutation was a point mutation (c.G277A) in exon1 of the MKRN3 gene which leads to glycine (G) being substituted with serine (S) at condon 93 (p.Gly93Ser) (Figures 1B, 2A). Further Sanger sequencing exhibited that her father had the same mutation, and no MKRN3 gene mutation was found in her mother (Figure 1B).
Mutation of MKRN3 attenuated its autoubiquitination and degradation. (A) Schematic view of human MKRN3 protein mutation involved in this study. H, histidine; C, cysteine. (B) Ubiquitination of the wild-type MKRN3 protein was more significant than that of the disease-associated mutant (G93S) in HEK293T cells. Cells were transformed with plasmids encoding HA-Ub and Flag-tagged wild-type MKRN3, or MKRN3 (G93S) mutant. MKRN3 proteins were immunoprecipitated using anti-Flag beads followed by immunoblotting with anti-HA to detect ubiquitination signals. WT, wild-type. (C) The wild-type MKRN3 protein was less stable than the G93S mutant. Flag-tagged wild-type MKRN3 or G93S mutant were expressed in HEK293T cells. Cells were treated with CHX (100 ug/ml) at different time durations (0, 2, or 4 h) before harvest for immunoblotting analysis. Data are presented as mean +- SD, one-way ANOVA was used with the Bonferroni post-hoc test over three independent experiments. *P < 0.05, significant difference; **P < 0.01, very significant difference, over three independent experiments.
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F2
Her mutation was a point mutation (c.G277A) in exon1 of the MKRN3 gene which leads to glycine (G) being substituted with serine (S) at condon 93 (p.Gly93Ser) (Figures 1B, 2A). The point mutation (p.G93S) is near the C3H domain of the MKRN3 protein (Figure 2A). Our study found that the ubiquitination of wild-type MKRN3 protein was more significant than that of the disease-associated mutant (G93S) in HEK293T cells (Figure 2B), and protein stability detected by immunoblotting analysis indicated that wild-type MKRN3 was less stable than the G93S mutant (Figure 2C).
Mutation of MKRN3 attenuated its inhibition on GnRH1-related signaling. (A) Schematic diagrams for the construction of luciferase reporter vectors for the human GNRH1, KISS1, and TAC3 gene promoters. The indicated region of promoters was amplified and inserted into the pGL3-basic vector. The miniCMV promoter was inserted and acted as a negative control. (B-D) A luciferase reporter assay was used to detect the relative activities of (B) miniCMV, (C)
GNRH1, (D)
KISS1, and (E)
TAC3 promoters in HEK293T cells. HEK293T cells were transfected with the indicated plasmids and luciferase activities were detected. Data are presented as mean +- SD, one-way ANOVA was used with the Bonferroni post-hoc test over three independent experiments. (F) Mutation of MKRN3 attenuated its inhibition on GnRH1 levels in GT1-7 cells. GT1-7 cells were transfected with empty vector, wild-type MKRN3, or the G93S mutant. A total of 48 h later, the supernatant was collected and detected by ELISA. Data are presented as mean +- SD, one-way ANOVA was used with the Bonferroni post-hoc test over three independent experiments. *P < 0.05, significant difference; **P < 0.01, very significant difference, over three independent experiments.
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F3
To study the function of wild-type and mutant MKRN3, luciferase reporter vectors for human GNRH1, KISS1, and TAC3 gene promoters were constructed, and the miniCMV promoter acted as a negative control (Figure 3A). As showed in Figure 3B, MKRN3 showed little activity on the miniCMV promoter. Compared to wild-type MKRN3, the G93S mutant led to weaker suppression of the transcriptional activity of the GNRH1, KISS1, and TAC3 promoters, as revealed in Figures 3C-E. In GT1-7 cells that were derived from mouse hypothalamic GnRH-positive neurons, wild-type MKRN3 significantly repressed the protein level of GnRH1, while the G93S mutant lost this effect (Figure 3F).