PMC8591216_01
General view and radiographic imaging of the lesion. (A) The right hand of the patient with Maffucci's syndrome showed deformities due to multiple enchondromas and a superficial hemangioma. The red arrows show hemangioma as a soft spherical bulge in light blue. (B) X-ray images of the right ulnar and radial bones and multiple metacarpophalangeal bones of the right hand show multiple reduced bone densities and partial expansive bone destruction (C). The X-ray shows a relatively normal bone mass in the left arm. (D) MR results of the patient's right hand showed the phalanges are circular or oval, multilocular, well-defined, iso-intensity and hyperintensity on T2WI (white arrows) and the soft tissue around the phalanx presents dilatative, well-bounded hyperintensity on T2WI (red arrows). (E) X-ray images of the patient's upper body and enlarged right arm are shown in the film. (F) X-ray images of the patient's feet have the same radiographic appearance as those of the upper limbs. (G) X-ray image of cranial and maxillofacial bones showed no apparent abnormalities.
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The right hand had dangerous tumor-like deformities, fingers in this hand displayed deformity shape of the visible nodular and soft spherical bulge in light blue ( Figure 1A ), and the right upper arm was shortening ( Figure 1B ), but her left hand showed healthy appearance ( Figure 1C ). MR examinations showed that the phalanges of the fingers are circular or ovoid, with multi-locular, well-defined chondromatoid lesions and the soft tissue around the phalanx of the finger presents dilatative, well-bounded hyperintensity on T2WI.( Figure 1D ). X-ray examinations demonstrated that the bones of the right palm, fingers of the right hand, right ulna, both radius ( Figure 1B ), humerus, scapulas, head of the right seventh rib ( Figure 1E ) and metatarsals of both feet ( Figure 1F ) were characterized by irregular swelling and morphological abnormalities. Bones in the cranial and maxillofacial regions, however, had no apparent abnormalities ( Figure 1G ).
Histological examination of lesions. (A, B) Histological examination manifesting typical enchondroma in the patient's right-hand finger bones. The specific manifestation was microscopic appearance of lobular hyaline cartilage with uniform chondrocytes, heaps of chondrocytes, uniform nuclear size and not deep staining (C, D). Samples were collected from patient's right-hand finger bones displaying cavernous hemangioma in the enchondroma tissues. Microscopically, a large number of neoplastic vascular tissues can be seen in the middle of cartilage tissue. The blood vessels are thin-walled and dilated and filled with red blood cells.
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Histological analysis indicated typical enchondroma changes in sections originating from right finger bones ( Figures 2A, B ), and cavernous hemangioma changes were noted in the enchondroma tissues from the same finger ( Figures 2C, D ).
Confirmation of somatic nonsynonymous mutation of IDH1 (c. C394T) (A) and ERC2 (c. C925A) (B) by sequencing PCR clones. The fragment flanking the mutated nucleotide was amplified by PCR and then cloned into a pMD18-T vector. Twenty-one clones were randomly sequenced to confirm the mutations and their frequency. (Normal:Normal; Mu:Mutation).
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Indeed, these mutations were absent in the blood DNA, while approximately 33.3% of the clones were positive for the IDH1 R132C mutation ( Figure 3A ), and 19.0% of the clones were positive for the ECR2 L309I mutation ( Figure 3B ).
The IDH1 R132C mutation is in Ollier disease, while the ERC2 L309I mutation is absent. The IDH1 R132C mutation was detected in most of the patients with Ollier disease (51/69 patients), and the mean frequency of this mutation was 63.3% in total sequence readouts, but the ECR2 L309I mutation was absent in all patients with Ollier disease examined.
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Excitingly, the IDH1 R132C mutation was detected in most of the patients with Ollier disease (51/69 patients), and the mean frequency of this mutation was 63.3% in total sequence readouts, but the ECR2 L309I mutation was absent in all of the patients with Ollier disease ( Figure 4 ).
Impact of the IDH1 R132C mutation on chondrocyte migration. The mouse chondrogenic ATDC5 cells were transduced with IDH1 wt and IDH1 mu (R132C) lentiviruses and then induced into chondrocytes as described. ATDC5 cells transduced with empty lentiviruses served as a control. (A) Representative images of each group from typical scratch migration assays. (B) A bar graphic figure shows the results of 5 independent scratch migration assays conducted. (C) Representative images of each group from transwell migration assays. (D) A bar graph shows data collected from 3 independent transwell migration assays. (*P<0.05).
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ATDC5, into chondrocytes after transduction with lentiviruses expressing wild-type IDH1 (IDH1-wt) or the R132C mutant (IDH1-mu). As expected, chondrocytes transduced with the IDH1-mu viruses exhibited significantly higher migration capacity ( Figure 5 ) and proliferation capability ( Figure 6A ).
The effect of the IDH1 R132C mutation on chondrocyte growth. (A) Proliferation results of chondrocytes after staining with WST-8 (n = 3). (B) Representative results of each group for cell cycle analysis. (C) A bar graphic figure shows the results collected from 3 independent replications. The IDH1 mu transduced cells manifested a significantly higher capacity for G1-S phase transition. (*P<0.05).
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ATDC5, into chondrocytes after transduction with lentiviruses expressing wild-type IDH1 (IDH1-wt) or the R132C mutant (IDH1-mu). As expected, chondrocytes transduced with the IDH1-mu viruses exhibited significantly higher migration capacity ( Figure 5 ) and proliferation capability ( Figure 6A ). Cell cycle analysis revealed that the R132C mutation significantly promoted the G1-S phase transition ( Figures 6B, C ).
The effects of the ERC2 L309I mutation on endothelial angiogenesis. HUVECs were transfected with ERC2 wt and ERC2 mu (L309I) lentiviruses and then subjected to angiogenesis analysis. (A) Proliferation results by analysis of WST-8 fluorescence (n = 3). (B) Representative images for analysis of tubular formation. (C) Quantitative results for capillary-like tube formation (n = 3). (D) Representative images for transwell migration assays. (E) A bar graph displaying the data from 3 independent transwell migration assays. (*P<0.05).
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C ) and migration ( Figures 7D, E ). Collectively, these data suggest that the IDH1 R132C mutation combines with the ERC2 L309I mutation to cause the development of Maffucci's syndrome.
ERC2 L309I mutation enhances endothelial intracellular calcium concentration. HUVECs were transduced with ERC2 wt and ERC2 mu (L309I) lentiviruses. HUVECs transduced with empty lentiviruses served as a control. Fura-2/AM was next applied to the cultures, and the intracellular free calcium concentration was then assessed for red fluorescence after washing under a fluorescence microscope.
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HUVECs by staining with Fura-2/AM (Invitrogen, OR, USA), a dye used to measure intracellular free calcium. HUVECs transduced with ERC2-mu displayed significantly higher intracellular free calcium concentrations than cells transduced with ERC2-wt ( Figure 8 ).