Age-dependent demise of GNAS-mutated skeletal stem cells and "normalization" of fibrous dysplasia of bone

Abstract

We studied the role of somatic mosaicism in fibrous dysplasia of bone (FD) within the context of skeletal ("mesenchymal") stem cells by assessing the frequency of mutated colony forming unit-fibroblasts (CFU-Fs) from FD lesions, and in some cases, from unaffected sites, in a series of patients. There was a tight inverse correlation between the percentage mutant CFU-F versus age, suggesting demise of mutant stem cells caused by exuberant apoptosis noted in samples from young patients. In older patients, either partially or completely normal bone/marrow histology was observed. On in vivo transplantation, FD ossicles were generated only by cell strains in which mutant CFU-Fs were identified. Strains that lacked mutant CFU-F (but were mutation positive) failed to regenerate an FD ossicle. These data indicate that GNAS mutations are only pathogenic when in clonogenic skeletal stem cells. From these data, we have evolved the novel concept of "normalization" of FD. As a lesion ages, mutant stem cells fail to self-renew, and their progeny are consumed by apoptosis, whereas residual normal stem cells survive, self-renew, and enable formation of a normal structure. This suggests that activating GNAS mutations disrupt a pathway that is required for skeletal stem cell self-renewal.

Figure FIG. 1.

Mutational analyses of non‐clonal and clonal cultures of BMSCs derived from fibrous dysplastic lesions. Non‐clonal cultures were analyzed by selective amplification of the mutated allele using a PNA primer to block amplification of the normal allele (which detects 1:200 mutated cells) and by direct DNA sequencing (which detects 1:2 or 3 mutated cells) to approximate the mutational load within the BMSC population. Clonal cultures were established to determine the colony forming efficiency (the number of CFU‐Fs), and isolated clones were analyzed by direct DNA sequencing to determine the number of mutated clones.

Figure FIG. 2.

Identification of mutations by PNA‐inhibited amplification (A‐C, 3′‐5′ strand sequenced) and direct DNA sequencing (D‐F, 5′‐3′ strand sequenced) (MW, molecular weight markers; +CON, positive control [DNA isolated from a mutant clone]; −CON, negative control [DNA isolated from a normal clone]). In some cultures of BMSCs derived from affected tissue, mutation (in this case, R201H) could be detected by both methods (A and D). In other cultures of BMSCs derived from affected tissue, mutation was detectable by PNA‐inhibited amplification (in this case, R201C) (B), but no mutation was found by direct DNA sequencing (E). BMSCs were also derived from clinically defined normal bone. In some cases, mutation was detected by PNA‐inhibited amplification (C), but no mutation was detected by direct DNA sequencing (F).

Figure FIG. 3.

Colony‐forming efficiency (CFE) and frequency of mutated clones. (A) Colony‐forming efficiency of single cell suspensions of bone marrow nucleated cells was determined for a series of normal donors (age range, 1–76 yr) from patients in which high levels of mutation were detected (age range, 7–35 yr) and from patients in which low levels of mutation were detected (age range, 35–52 yr). The average CFE was ∼18‐fold higher in samples from patients with a high level of mutation compared with that of normal donors (*p = 0.0001) and significantly higher compared with that of patients with a low level of mutation (^# p = 0.045). There was no statistical difference between samples from normal donors compared with those from patients with a low level of mutation. (B) Clones (∼20 from each patient) isolated from low‐density cultures of single cell suspensions of bone marrow mononuclear cells were genotyped by direct DNA sequencing. The percent of mutated clones (representative of the percent of mutated CFU‐Fs) was compared with the age of the patient by linear regression and was found to have a tight negative correlation.

Figure FIG. 4.

Bone scintigraphy (A), X‐ray images (B). and histology of FD lesions (C) in three different patients >35 yr of age. Whereas both scintigraphy (panels 1–3) and X‐ray images (panels 4–6) show the presence of lesions, the histology of putatively lesional bone shows normal histology in two of three patients. In one patient, histology shows normal bone and hematopoietically active marrow, consistent with the histology of the iliac crest in a normal adult subject (panel 7). In the second patient, histology shows normal bone trabeculae and adipose marrow, consistent with the histology of the femur in a normal adult (panel 8). In the third patient, nondescript fibrous tissue is seen along with normal bone trabeculae (panel 9). The trabeculae showed normal lamellar structure, instead of the woven texture typical of FD, on examination by polarized light microscopy (data not shown).

Figure FIG. 5.

Histological changes in the “normalization” of fibrous dysplastic lesions. von Kossa and Giemsa (A and B) and H&E (C and D) staining of lesional tissue from patient 7 (18 yr old) with high mutation levels (A and C) and from patient 10 (sample b, 35 yr old) with low mutation levels (B and D). In patient 7, bone is poorly mineralized, and there is excessive osteoid (* in A). Osteoblasts display a distinctive retracted, stellate morphology (white arrows in A, black arrows in C), characteristics that are typically found in FD lesions. In patient 10, mineralization is normal (B), and normal‐appearing cuboidal osteoblasts cover the bone‐forming surfaces (white arrows in B, black arrows in D).

Figure FIG. 6.

Apoptosis in fibrous dysplastic tissue. (A) TUNEL analysis of tissue from patients with typical FD histology and high mutational load shows extremely high frequencies of apoptotic cells throughout the fibrotic marrow (FT) and bone (B). (B) High levels of TUNEL⁺ cells are found on the bone surfaces (arrow) and within the abnormal bone (arrowhead). (C–E) Apoptotic bodies in nondecalcified sections stained with von Kossa and Giemsa (white arrows). (F–I) Apoptotic bodies in decalcified sections stained with H&E (black arrows).

Figure FIG. 7.

Transplantation of BMSCs in vivo. BMSCs derived from FD lesions and normal sites were combined with HA/TCP as a carrier (C) and transplanted subcutaneously into immunocompromised mice. (A) BMSCs from lesions with high levels of mutation (patient 1) reproduced a fibrous dysplastic ossicle characterized by formation of poorly mineralized woven bone (WB) and fibrous tissue (FT), reminiscent of a native lesion. (B) BMSCs from lesions in which the level of mutation was only detectable by PNA‐inhibited amplification (patient 10, sample b) formed normal lamellar bone (LB) and established a normal hematopoietic marrow (HP). (C) BMSCs derived from a normal site, but mutation positive by the PNA assay (patient 16, sample a) also established a normal ossicle.