Alopecia, neurological defects, and endocrinopathy syndrome caused by decreased expression of RBM28, a nucleolar protein associated with ribosome biogenesis

Abstract

Single-gene disorders offer unique opportunities to shed light upon fundamental physiological processes in humans. We investigated an autosomal-recessive phenotype characterized by alopecia, progressive neurological defects, and endocrinopathy (ANE syndrome). By using homozygosity mapping and candidate-gene analysis, we identified a loss-of-function mutation in RBM28, encoding a nucleolar protein. RBM28 yeast ortholog, Nop4p, was previously found to regulate ribosome biogenesis. Accordingly, electron microscopy revealed marked ribosome depletion and structural abnormalities of the rough endoplasmic reticulum in patient cells, ascribing ANE syndrome to the restricted group of inherited disorders associated with ribosomal dysfunction.

Figure 1

Clinical Features of ANE Syndrome Patients display varying patterns of alopecia including (A) alopecia universalis, (B) hypotrichosis, (C) near-normal scalp hair associated with absence of body and (D) axillary hair; additional features include (E) gynecomastia, (F) flexural reticulate hyperpigmentation and (G) hypodontia and tooth malplacement; (H) cerebral MRI (T1 weighted after gadolinium injection) shows normal hypothalamus, hypoplastic thin anterior pituitary gland at the bottom of the sella turcica (thick arrow), and ectopic posterior pituitary hyperintense signal located at a proximal level of the pituitary stalk (thin arrow); (I) a skin biopsy obtained from patient scalp reveals absence of mature hair follicles, and instead, presence of dermal cysts (Cy) as well as elongated vertically oriented epithelial structures associated with sebaceous glands, corresponding to rudimentary hair follicles (Rhf).

Figure 2

Genetic Mapping of ANE Syndrome (A) Multipoint LOD score analysis in ANE syndrome. The LOD scores are plotted against the physical distances separating the markers assessed. (B) Haplotype analysis of the ANE family with polymorphic markers on chromosome 7q31.32-7q32 reveals a homozygous haplotype shared by all patients (boxed in red).

Figure 3

Mutation Analysis (A) Sequence analysis of RBM28 reveals a homozygous T→C transition at cDNA position 1052 of the RBM28 gene (red arrow, upper panel). The wild-type sequence is given for comparison (lower panel). (B) PCR-RFLP analysis confirms segregation of the mutation in the family. PCR amplification was performed as described in the text. Mutation c.T1052C creates a recognition site for BSAJI. Thus, affected patients display two 127 bp and 132 bp fragments (appearing as one band) and healthy individuals show a 257 bp fragment only, whereas all fragments are found in heterozygous carriers of the mutation.

Figure 4

Tissue Expression of RBM28 RBM28 gene expression was assessed with Clontech tissue blot cDNA array. The tissue used in each lane is as follows: lane 1, trachea; lane 2, thymus; lane 3, thyroid; lane 4, testes; lane 5, spleen; lane 6, muscle; lane 7, small intestine; lane 8, prostate; lane 9, heart; lane 10, ovary; lane 11, cervix; lane 12, bladder; lane 13, colon; lane 14, brain; lane 15, lungs; lane 16, liver; lane 17, kidney; lane 18, esophagus; lane 19, adipose tissue; and lane 20, placenta. Expression of RBM28 was compared to that of ACTB.

Figure 5

Consequences of p.L351P Mutation in RBM28 (A) ClustalW analysis of the RBM28 protein region encompassing the mutation site demonstrates that L351 (boxed in red) is conserved across species. (B) The sequence of the N-terminal part of RBM28 was modeled with the Phyre homology recognition engine server with the crystal structure of the N-terminal region of the yeast RNA splicing factor Prp24 (PDB code 2GHP). Four hundred and twenty residues could be modeled in this fashion with an extremely high degree of confidence (E = 10⁻²⁴). The Prp24 structure includes three RNA recognition motifs (RRM), consisting of a four-stranded β sheet flanked by two α helices. Wild-type L351 (white ellipse, left panel) is located on the C-terminal end of helix 1 of RRM3 and is expected to destabilize the protein α-helix 1 when mutated to proline (white ellipse, right panel). (C) Protein was extracted from fibroblast cell cultures established from two ANE patients (P1 and P2) and a control healthy individual (C) and immunoblotted against anti-RBM28 antibodies. Membranes were reblotted with anti-β-actin antibodies to control for protein loading. (D) Cultured fibroblasts obtained from a patient (lower panel) and a control individual (upper panel) were stained with an anti-RBM28 antibody (green) and propidium iodide (red) and were examined with confocal microscopy. Merged images are presented. Note increased focal expression of RBM28 in the nucleus of control cells and markedly decreased expression of the protein in patient cells. (E) Skin-biopsy sections obtained from a patient (right panel) and a control individual (left panel) were stained with antibodies directed against RBM28. Immunostaining is markedly decreased in the skin of the patient as compared with control skin (original magnification, 400×).

Figure 6

Ultrastructural Abnormalities Associated with RBM28 Deficiency (A) Transmission electron microscopy (TEM) of cultured fibroblasts in a patient (right panels) and a healthy control individual (left panels) revealed dilated cisterns of the rough endoplasmic reticulum (RER, upper panels) and decreased number of free ribosomes (red circles, lower panels) in the patient cells. The rectangles in the upper panels correspond to the magnified electron micrographs shown in the lower panels. (B) Free cytoplasmic ribosomes were counted at a TEM magnification of 40000× in control and patient fibroblasts in six independent samples (>1500 ribosomes counted in each group). Results are given as number of free ribosomes per μm² ± SE. Statistical differences were assessed with a paired Student's t test.