Mutations in C2orf37, encoding a nucleolar protein, cause hypogonadism, alopecia, diabetes mellitus, mental retardation, and extrapyramidal syndrome

Abstract

Hypogonadism, alopecia, diabetes mellitus, mental retardation, and extrapyramidal syndrome (also referenced as Woodhouse-Sakati syndrome) is a rare autosomal recessive multisystemic disorder. We have identified a founder mutation consisting of a single base-pair deletion in C2orf37 in eight families of Saudi origin. Three other loss-of-function mutations were subsequently discovered in patients of different ethnicities. The gene encodes a nucleolar protein of unknown function, and the cellular phenotype observed in patient lymphoblasts implicates a role for the nucleolus in the pathogenesis of this disease. Our findings expand the list of human disorders linked to the nucleolus and further highlight the developmental and/or maintenance functions of this organelle.

Figure 1

C2orf37 Mutations Are Responsible for Woodhouse-Sakati Syndrome (A) WSS patient with dystonia and alopecia. (B) One illustrative WSS pedigree showing multiple consanguinity loops. (C) Haplotype analysis of Saudi WSS patients. Each column represents one of the 18 patients studied, and each row represents one of the SNPs used for the analysis. Color codes specify the genotypes of each SNP as AA, BB, or AB, and the genetic distance of each SNP from C2orf37 is listed to the right. (D) Critical linkage interval obtained after fine mapping, with C2orf37 in red. The two major isoforms are shown as well, with open boxes representing untranslated regions and shaded boxes representing the coding sequences (not drawn to scale). The ORFs are in-frame and utilize the same stop codon. Chromatograms of the four mutations show control sequences on top, with mutations denoted by asterisks. Arrowheads indicate location within the gene. RT-PCR analysis (using F1 and R1 primers) of a normal control and one WSS patient from (4) reveals skipping of exon 10 in the latter.

Figure 2

Immunohistochemistry and In Situ Hybridization on Mouse Embryonic Tissues Intense nucleolar staining of hepatocytes (heart tissue is visible in the upper right corner) at E16.5 (A), brain cells at E12.5 (B), and different layers of skin including a hair follicle (inset) at E14.5 (C). Bottom panels present in situ hybridization patterns observed for liver tissue at E13.5 (D), cortex of the brain at E15.5 (E), and skin at E15.5 (F).

Figure 3

Nucleolar Staining of C2orf37 Is Specific (A) Immunohistochemistry on paraffin sections of a HEK293 cell block showing strong nucleolar and weak cytoplasmic staining. (B) Nucleolar but not cytoplasmic staining is abrogated when the slide is incubated with the peptide antigen prior to staining with C2orf37 antibody. (C) Buffer negative control, with the primary antibody excluded.

Figure 4

Both Major Isoforms of C2orf37 Localize to the Nucleolus in Tissue Culture (A–C) Overexpression of the β-isoform protein from a cloned expression construct results in nucleolar localization of the peptide in HEK293 cells. The same pattern appears for the α-isoform. (D–F) Endogenous C2orf37 colocalizes with B23, a nucleolar marker, in lymphoblasts. (G–L) C2orf37 and B23 remain largely nucleolar after low-dose actinomycin D treatment of control lymphoblasts (G–I), but largely nucleoplasmic in patient lymphoblasts (J–L), where their colocalization is partially lost (indicated by arrowhead).

Figure 5

Nucleolar Localization of C2orf37 in HEK293 Cells Is Less Dependent than B23 on Active Transcription (A–E) C2orf37 and B23 colocalize at the nucleolus under normal conditions in HEK293 cells. (F–J) Low-dose actinomycin D treatment results in full displacement of B23 but only partial displacement of C2orf37 to the nucleoplasm (arrowheads point to foci of retained nucleolar localization). (K–O) High-dose actinomycin D results in a similar but more potent effect, given that less foci of retained C2orf37 nucleolar localization can be seen.