DLG2 variants in patients with pubertal disorders

Abstract

Purpose: Impaired function of gonadotropin-releasing hormone (GnRH) neurons can cause a phenotypic spectrum ranging from delayed puberty to isolated hypogonadotropic hypogonadism (IHH). We sought to identify a new genetic etiology for these conditions.

Methods: Exome sequencing was performed in an extended family with autosomal dominant, markedly delayed puberty. The effects of the variant were studied in a GnRH neuronal cell line. Variants in the same gene were sought in a large cohort of individuals with IHH.

Results: We identified a rare missense variant (F900V) in DLG2 (which encodes PSD-93) that cosegregated with the delayed puberty. The variant decreased GnRH expression in vitro. PSD-93 is an anchoring protein of NMDA receptors, a type of glutamate receptor that has been implicated in the control of puberty in laboratory animals. The F900V variant impaired the interaction between PSD-93 and a known binding partner, Fyn, which phosphorylates NMDA receptors. Variants in DLG2 that also decreased GnRH expression were identified in three unrelated families with IHH.

Conclusion: The findings indicate that variants in DLG2/PSD-93 cause autosomal dominant delayed puberty and may also contribute to IHH. The findings also suggest that the pathogenesis involves impaired NMDA receptor signaling and consequently decreased GnRH secretion.

Keywords: DLG2; NMDA receptors; PSD-93; hypogonadotropic hypogonadism; puberty.

Figure 1.. A family with markedly delayed puberty.

A. Pedigree. Arrow indicates proband. Closed symbol, delayed puberty; open symbol, normal timing of puberty; F900V, a variant in DLG2/PSD-93; WT, wild-type DLG2/PSD-93.

Figure 2.. Effect of F900V DLG2/PSD-93 variant on Gnrh1 expression in GT1–7 cells

In GT1–7 cells (between 13–15 passages in culture), wild-type (WT) PSD-93 expression stimulated Gnrh1 expression compared to empty vector (EV) for both mRNA (normalized to 18s rRNA, Panel A, P<0.001, n=10) and GnRH peptide expression (normalized to total protein, Panel B, P<0.001, n=14). The F900V PSD-93 mutant impaired this stimulation both in mRNA levels (P=0.003, Panel A) and intracellular peptide levels (P=0.008, Panel B).

Figure 3.

A and B) Co-immunoprecipitation of PSD-93 and myc-Fyn in HEK293T cells. Fyn tagged with myc and either wild-type or F900V mutant PSD-93 were co-expressed in HEK293T cells and immunoprecipitated with antibody against PSD-93. The input protein (prior to immunoprecipitation) and immunoprecipitated protein were analyzed by western blot (3A) using antibody against the myc tag. The immunoprecipitated myc-Fyn bands were quantified and normalized to input (3B). The findings indicate that the variant in PSD-93 decreased binding to Fyn (n=3, P=0.005). WT, wild-type; F-V, F900V variant; IP, immunoprecipitation. C and D) Phosphorylation of GluN2B. Fyn, GluN1, GluN2B, and either wild-type or F900V PSD-93 were co-expressed in HEK293T cells and the phosphorylation of GluN2B at Y1472 was assessed by western blot. The F900V variant decreased phosphorylation (n=3). ***; P = 0.0004, *; P = 0.01.

Figure 3.

A and B) Co-immunoprecipitation of PSD-93 and myc-Fyn in HEK293T cells. Fyn tagged with myc and either wild-type or F900V mutant PSD-93 were co-expressed in HEK293T cells and immunoprecipitated with antibody against PSD-93. The input protein (prior to immunoprecipitation) and immunoprecipitated protein were analyzed by western blot (3A) using antibody against the myc tag. The immunoprecipitated myc-Fyn bands were quantified and normalized to input (3B). The findings indicate that the variant in PSD-93 decreased binding to Fyn (n=3, P=0.005). WT, wild-type; F-V, F900V variant; IP, immunoprecipitation. C and D) Phosphorylation of GluN2B. Fyn, GluN1, GluN2B, and either wild-type or F900V PSD-93 were co-expressed in HEK293T cells and the phosphorylation of GluN2B at Y1472 was assessed by western blot. The F900V variant decreased phosphorylation (n=3). ***; P = 0.0004, *; P = 0.01.

Figure 4.. Three unrelated families with variants in DLG2/PSD-93.

Family A. Two siblings with unaffected parents presented with normosmic hypogonadotropic hypogonadism. Each sibling was heterozygous for an E140K variant in DLG2/PSD-93. Neither DNA nor a pubertal history was available for the father of the affected individuals. Family B. Two siblings with unaffected parents presented with Kallmann syndrome. Both siblings and the unaffected father were heterozygous for a I901V variant in DLG2/PSD-93. Family C. A patient presented with Kallmann syndrome. She and her father with delayed puberty were heterozygous for a Q166H variant in DLG2. Below each pedigree is shown the results of in vitro studies of the specific Dlg2/PSD-93 variant found in that family. Empty vector (EV), wild-type (WT) Dlg2/PSD-93 and mutant Dlg2/PSD-93 were expressed in GT1–7 cells and Gnrh1 mRNA expression was measured. The three Dlg2/PSD-93 variants suppressed the stimulation of Gnrh1 compared to wild-type Dlg2/PSD-93 (n=8). Arrow indicates probands. Left upper black quadrant, IHH; right lower black quadrant, anosmia; right upper black quadrant, delayed puberty.