Loss-of-function mutations in IGSF1 cause an X-linked syndrome of central hypothyroidism and testicular enlargement

Abstract

Congenital central hypothyroidism occurs either in isolation or in conjunction with other pituitary hormone deficits. Using exome and candidate gene sequencing, we identified 8 distinct mutations and 2 deletions in IGSF1 in males from 11 unrelated families with central hypothyroidism, testicular enlargement and variably low prolactin concentrations. IGSF1 is a membrane glycoprotein that is highly expressed in the anterior pituitary gland, and the identified mutations impair its trafficking to the cell surface in heterologous cells. Igsf1-deficient male mice show diminished pituitary and serum thyroid-stimulating hormone (TSH) concentrations, reduced pituitary thyrotropin-releasing hormone (TRH) receptor expression, decreased triiodothyronine concentrations and increased body mass. Collectively, our observations delineate a new X-linked disorder in which loss-of-function mutations in IGSF1 cause central hypothyroidism, likely secondary to an associated impairment in pituitary TRH signaling.

Figure 1. IGSF1 mutations identified in patients with central hypothyroidism

(a) Pedigree of family A. Small horizontal lines signify that the mutation was confirmed. (b) Pedigree of family B. (c) Schematic representation of IGSF1 protein domain structure and relative positions of identified mutations.

Figure 2. IGSF1 is expressed in anterior pituitary gland

(a) Expression of IGSF1/Igsf1 mRNA in murine embryonic day 12.5 and human embryo Carnegie stage 18 Rathke’s pouch progenitors as detected by in situ hybridization. Scale bars, 10 μm. (b) Immunofluorescence using IGSF1-CTD antibody and antibodies against the indicated anterior pituitary hormones (TSH: thyrotropes; GH: somatotropes; prolactin: lactotropes; LH: gonadotropes) was performed in WT E18.5 mouse pituitary. Scale bars, 10 μm.

Figure 3. Mutations in IGSF1 impair its plasma membrane trafficking

(a) HEK293 cells were transfected with pcDNA3 (empty vector) or the indicated wild-type or mutant IGSF1 expression vectors. Protein lysates were deglycosylated with either PNGaseF (P) or EndoH (E), resolved by SDS-PAGE, and immunoblotted using an IGSF1-CTD antibody. Non-specific bands are indicated by *. (b) HEK293 cells were transfected with the same constructs as in (a). Expression of IGSF1-CTD was analyzed by immunofluorescence using the IGSF1-CTD antibody under non-permeabilizing and permeabilizing conditions. Nuclei were stained with DAPI (blue). Scale bars, 10 μm. (c) HEK293 cells were transfected with pcDNA3 or the indicated wild-type or mutant IGSF1 expression vectors. Membrane expression of IGSF1-CTD was analyzed by cell-surface biotinylation.

Figure 4. Igsf1^Δex1 mice have several characteristics of central hypothyroidism

(a) Pituitary Tshb mRNA levels in 12-week old wild-type and Igsf1^Δex1 mice (N=6/genotype). (b) Pituitary TSH content in male wild-type and Igsf1^Δex1 mice (N=6/genotype). (c) Serum TSH levels in adult wild-type and Igsf1^Δex1 mice(N=6/genotype). (d) Serum total T3 levels in adult wild-type and Igsf1^Δex1 mice (N=14-16/genotype). (e) Trhr mRNA levels in 12-week old wild-type and Igsf1^Δex1 mice (N=6/genotype). (f) Trh mRNA levels in 12-week old wild-type and Igsf1^Δex1 mice (N=5/genotype). Statistical significance was determined by two-tailed Student’s t-test in each panel.