Impaired osteoblast and osteoclast function characterize the osteoporosis of Snyder - Robinson syndrome

Abstract

Background: Snyder-Robinson Syndrome (SRS) is an X-linked intellectual disability disorder also characterized by osteoporosis, scoliosis, and dysmorphic facial features. It is caused by mutations in SMS, a ubiquitously expressed gene encoding the polyamine biosynthetic enzyme spermine synthase. We hypothesized that the tissue specificity of SRS arises from differential sensitivity to spermidine toxicity or spermine deficiency.

Methods: We performed detailed clinical, endocrine, histopathologic, and morphometric studies on two affected brothers with a spermine synthase loss of function mutation (NM_004595.4:c.443A > G, p.Gln148Arg). We also measured spermine and spermidine levels in cultured human bone marrow stromal cells (hBMSCs) and fibroblasts using the Biochrom 30 polyamine protocol and assessed the osteogenic potential of hBMSCs.

Results: In addition to the known tissue-specific features of SRS, the propositi manifested retinal pigmentary changes, recurrent episodes of hyper- and hypoglycemia, nephrocalcinosis, renal cysts, and frequent respiratory infections. Bone histopathology and morphometry identified a profound depletion of osteoblasts and osteoclasts, absence of a trabecular meshwork, a low bone volume and a thin cortex. Comparison of cultured fibroblasts from affected and unaffected individuals showed relatively small changes in polyamine content, whereas comparison of cultured osteoblasts identified marked differences in spermidine and spermine content. Osteogenic differentiation of the SRS-derived hBMSCs identified a severe deficiency of calcium phosphate mineralization.

Conclusions: Our findings support the hypothesis that cell specific alterations in polyamine metabolism contribute to the tissue specificity of SRS features, and that the low bone density arises from a failure of mineralization.

Figure 1

Clinical and radiographic features. A. Face of patient II-1. B. Face of patient II-3. C. Palate of patient II-1. D. Hands of patient II-3. E-J. Skeletal radiographs of Patient II-1 showing the left humerus (E), left forearm (F), left hand (G), pelvis (H), left femur (I) and left lower leg (J). Note the gracile bones and undermineralization as well as the healing humeral fracture (E).

Figure 2

Segregation, mutational analysis, and functional consequences of a novel SMS variant. A. Pedigree of the family of the propositi. Affected males are shown by black squares. B. Sanger sequencing chromatograms showing the segregation of the SMS mutation NM_004595.4:c.443A > G from the carrier mother to the affected boys. The unaffected father did not have this mutation. C. Conservation of the p.Gln148 (p.Q148) residue across species. D. Drawing of the human SMS protein crystal complexed with spermidine and 5-methylthioadenosine. The mutated amino acid (Gln148) is highlighted in yellow [Mac PyMOL [23]]. E-J. Immunofluorescent detection of SMS protein subcellular distribution in unaffected (E, F), Patient II-1 (G, H) and Patient II-3 (I, J) skin fibroblasts. SMS protein is shown in red and the nucleus is shown in blue. K. Immunoblot of skin fibroblast lysates showing reduced SMS protein levels in the patients (II-1, II-3) compared to an unaffected control (cnt). Tubulin is shown as a loading control. L. Graph showing steady state SMS protein levels in the patient and control fibroblasts relative to ß-tubulin levels. The data are based on 3 independent experiments for each cell line. M. Graph quantifying immunoblot detected steady state SMS protein levels in the cytoplasm and nuclei of patient and control fibroblasts. The cytoplasmic expression was normalized to β-tubulin expression and the nuclear expression to p84 expression. The data are based on 2 independent experiments for each cell line. N. SMS enzyme activity (spermidine d8 peak per hour) in lymphoblasts of unaffected individuals (Cnt), a cohort of 4 individuals with SRS (SRS) and patient II-1, * p < 0.05.

Figure 3

Characterization of the bone and osteoblast pathology. A. Photograph of the bone biopsy. B. Steady state SMS mRNA levels relative to GAPDH expression in cultured fibroblasts and osteoblasts. The patient’s cells did not differ significantly from controls. Data were derived by qRT-PCR analysis of 3 independent extractions of total RNA. C. Immunoblot showing steady state SMS protein expression in patient and control osteoblasts. ß-tubulin is shown as a loading control. D. Graph showing steady state SMS protein levels in the patient and control hBMSCs relative to ß-tubulin levels; there was no significant difference. The data are based on 3 independent experiments for each cell line. E-J. Immunofluorescent detection of SMS protein subcellular distribution in unaffected (E-G) and Patient II-1 (H-J) hBMSCs. SMS protein is shown in red and the nucleus is shown in blue. K. Graph quantifying immunoblot detected steady state SMS protein levels in the cytoplasm and nuclei of patient and control hBMSCs. The cytoplasmic expression was normalized to β-tubulin expression and the nuclear expression to p84 expression. L. Polyamine quantification in fibroblasts and osteoblasts. Note that the patient hBMSCs have a more striking imbalance of spermidine and spermine levels than do the patient fibroblasts, * p < 0.05, *** p < 0.005. M. Osteogenic potential of bone marrow stromal cells (hBMSCs) isolated from Patient II-1 sample is markedly lower than that of an unaffected control (cnt). The hBMSCs were seeded in triplicates (6x10⁴/12-well) and either kept untreated (-) or treated (+) with osteogenic differentiation media (see Methods) for 18 days. After the treatment, cells were fixed and were stained with Alizarin Red S to check for calcium deposition, a marker of osteogenic differentiation.