Analysis of Anthrax 2 Expression in Gingiva and Skin

BACKGROUND: Juvenile Hyaline Fibromatosis (JHF) and Infantile Systemic Hyalinosis (ISH), rare autosomal recessive disorders due to ANTXR2 mutations, are characterized clinically by subcutaneous tumors, gingival hypertrophy, joint contractures and osteolytic lesions. Histologically, deposition of hyaline material is pathognomonic. We have previously identified a founder mutation, c.1074delT, in 4 JHF probands.

OBJECTIVE: To understand why gingival hypertrophy is associated with ANTXR2 mutations.

METHODS: ANTXR2 expression in normal and JHF gingival fibroblasts and keratinocytes was determined by Taqman assays. Isoforms ANTXR2-489, ANTXR2-488, ANTXR2-386, and ANTXR2-322 were evaluated using RT-PCR primers designed to amplify each isoform. Immunohistochemical evaluations of JHF and control gingiva were performed with antibodies for ANTXR2, collagen IV, laminin á5 and MMP-9.

RESULTS: Taqman assay 2 (detects all 4 isoforms) gave higher expression levels than assay 1 (detects ANTXR2-489 and -488). Higher levels were seen in cultured samples compared to the uncultured tissues with both assays. JHF samples had low levels of expression, probably reflecting decreased transcription or decreased stability of mutant mRNA. By RT-PCR, the predominant isoform in all samples was ANTXR2-488. ANTXR2-322 was expressed at levels significantly lower (at least 30 times) than ANTXR2-488 and ANTXR2-489. The ANTXR2-386 isoform was not expressed in any tissue. JHF tissues demonstrated anomalies in the number and development of vascular cells. Immunohistochemical findings demonstrate immature collagen fibrils and localization of ANTXR2 in immature sprouting endothelial cells in the JHF stroma.

CONCLUSION: The Taqman and RT-PCR studies suggest the possibility that other ANTXR2 isoforms may be present. Further studies are needed to verify the presence of any other isoforms. JHF stroma are characterized by a large number of premature endothelial cells which fail to form mature blood vessels. The increased amounts of mutant ANTRX2 in immature blood vessels suggesting anomalies of angiogenesis are responsible for gingival hypertrophy in JHF.