Osteogenesis imperfecta (OI) is a genetic disorder of connective tissue characterized by bone fragility and alteration in synthesis and posttranslational modification of type I collagen. Autosomal dominant OI is caused by mutations in the genes (COL1A1 or COL1A2) encoding the chains of type I collagen. Bruck syndrome is a recessive disorder featuring congenital contractures in addition to bone fragility; Bruck syndrome type 2 is caused by mutations in PLOD2 encoding collagen lysyl hydroxylase, whereas Bruck syndrome type 1 has been mapped to chromosome 17, with evidence suggesting region 17p12, but the gene has remained elusive so far. Recently, the molecular spectrum of OI has been expanded with the description of the basis of a unique posttranslational modification of type I procollagen, that is, 3-prolyl-hydroxylation. Three proteins, cartilage-associated protein (CRTAP), prolyl-3-hydroxylase-1 (P3H1, encoded by the LEPRE1 gene), and the prolyl cis-trans isomerase cyclophilin-B (PPIB), form a complex that is required for fibrillar collagen 3-prolyl-hydroxylation, and mutations in each gene have been shown to cause recessive forms of OI. Since then, an additional putative collagen chaperone complex, composed of FKBP10 (also known as FKBP65) and SERPINH1 (also known as HSP47), also has been shown to be mutated in recessive OI. Here we describe five families with OI-like bone fragility in association with congenital contractures who all had FKBP10 mutations. Therefore, we conclude that FKBP10 mutations are a cause of recessive osteogenesis imperfecta and Bruck syndrome, possibly Bruck syndrome Type 1 since the location on chromosome 17 has not been definitely localized. © 2011 American Society for Bone and Mineral Research.

Osteogenesis imperfecta (OI) is a spectrum of genetic disorders characterized by bone fragility. It is caused by dominant mutations affecting the synthesis and/or structure of type I procollagen or by recessively inherited mutations in genes responsible for the posttranslational processing/trafficking of type I procollagen. Recessive OI type VI is unique among OI types in that it is characterized by an increased amount of unmineralized osteoid, thereby suggesting a distinct disease mechanism. In a large consanguineous family with OI type VI, we performed homozygosity mapping and next-generation sequencing of the candidate gene region to isolate and identify the causative gene. We describe loss of function mutations in serpin peptidase inhibitor, clade F, member 1 (SERPINF 1) in two affected members of this family and in an additional unrelated patient with OI type VI. SERPINF1 encodes pigment epithelium-derived factor. Hence, loss of pigment epithelium-derived factor function constitutes a novel mechanism for OI and shows its involvement in bone mineralization. © 2011 American Society for Bone and Mineral Research

Tricho-rhino-phalangeal syndrome (TRPS) is an autosomal dominant craniofacial and skeletal dysplasia that is caused by mutations involving the TRPS1 gene. Patients with TRPS have short stature, hip abnormalities, cone-shaped epiphyses and premature closure of growth plates reflecting defects in endochondral ossification. The TRPS1 gene encodes for the transcription factor TRPS1 that has been demonstrated to repress transcription in vitro. To elucidate the molecular mechanisms underlying skeletal abnormalities in TRPS, we analyzed Trps1 mutant mice (Trps1DeltaGT mice). Analyses of growth plates demonstrated delayed chondrocyte differentiation and accelerated mineralization of perichondrium in Trps1 mutant mice. These abnormalities were accompanied by increased Runx2 and Ihh expression and increased Indian hedgehog signaling. We demonstrated that Trps1 physically interacts with Runx2 and represses Runx2-mediated trans-activation. Importantly, generation of Trps1(DeltaGT/+);Runx2(+/-) double heterozygous mice rescued the opposite growth plate phenotypes of single mutants, demonstrating the genetic interaction between Trps1 and Runx2 transcription factors. Collectively, these data suggest that skeletal dysplasia in TRPS is caused by dysregulation of chondrocyte and perichondrium development partially due to loss of Trps1 repression of Runx2.

The type X collagen gene (Col10a1) is a specific molecular marker of hypertrophic chondrocytes during endochondral bone formation. Mutations in human COL10A1 and altered chondrocyte hypertrophy have been associated with multiple skeletal disorders. However, until recently, the cis-enhancer element that specifies Col10a1 expression in hypertrophic chondrocytes in vivo has remained unidentified. Previously, we and others have shown that the Col10a1 distal promoter (24.4 to 23.8 kb) may harbor a critical enhancer that mediates its tissue specificity in transgenic mice studies. Here, we report further localization of the cis-enhancer element within this Col10a1 distal promoter by using a similar transgenic mouse approach. We identify a 150-bp Col10a1 promoter element (24296 to 24147 bp) that is sufficient to direct its tissue-specific expression in vivo. In silico analysis identified several putative transcription factor binding sites including two potential activator protein-1 (AP-1) sites within its 59-and 39-ends (24276 to 24243 and 24166 to 24152 bp), respectively. Interestingly, transgenic mice using a reporter construct deleted for these two AP-1 elements still showed tissue-specific reporter activity. EMSAs using oligonucleotide probes derived from this region and MCT cell nuclear extracts identified DNA/protein complexes that were enriched from cells stimulated to hypertrophy. Moreover, these elements mediated increased reporter activity on transfection into MCT cells. These data define a 90-bp cis-enhancer required for tissuespecific Col10a1 expression in vivo and putative DNA/protein complexes that contribute to the regulation of chondrocyte hypertrophy. This work will enable us to identify candidate transcription factors essential both for skeletal development and for the pathogenesis of skeletal disorders.

We have recently shown that a 150-bp Col10a1 distal promoter (−4296 to −4147 bp) is sufficient to direct hypertrophic chondrocyte-specific reporter (LacZ) expression in vivo. More recently, through detailed sequence analysis we identified two putative tandem-repeat Runx2 binding sites within the 3′-end of this 150-bp region (TGTGGG-TGTGGC, −4187 to −4176 bp). Candidate electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation, and transfection studies demonstrate that these putative Runx2 sites bind Runx2 and mediate upregulated Col10a1/reporter activity in vitro. Transgenic studies using the 5′-sequence without Runx2 sites were not able to drive the cell-specific LacZ reporter activity, suggesting the in vivo requirement of the Runx2 sites located in the 3′-end in mediating Col10a1/reporter expression. Indeed, mutating the Runx2 sites in the context of the 150-bp promoter abolishes its capacity to drive hypertrophic chondrocyte-specific reporter expression in transgenic mice. We have also generated multiple transgenic mouse lines using only the 3′-sequence containing the Runx2 sites to drive the LacZ gene. Interestingly, no hypertrophic chondrocyte-specific blue staining was observed in these transgenic mice. Together, our data support that Runx2 directly interacts with murine Col10a1 cis-enhancer. This interaction is required but not sufficient for cell-specific Col10a1 promoter activity in vivo. Additional cooperative/repressive elements within the 5′- or 3′-sequences of this 150-bp promoter are needed to work with Runx2 together to mediate cell-specific Col10a1 expression. Further delineation of these elements/factors has the potential to identify novel therapeutic targets for multiple skeletal disorders, including osteoarthritis, that show abnormal Col10a1 expression and altered chondrocyte maturation. © 2011 American Society for Bone and Mineral Research

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Tricho-rhino-phalangeal syndromes (TRPS) are caused by mutation or deletion of TRPS1, a gene encoding a GATA transcription factor. These disorders are characterized by abnormalities of the hair, face, and selected bones. Rare cases of individuals with TRPS displaying supernumerary teeth have been reported, but none of these has been examined molecularly. We used two different approaches to investigate a possible role of TRPS1 during tooth development. We looked at the expression of Tprs1 during mouse tooth development and analyzed the craniofacial defects of Trps1 mutant mice. In parallel, we investigated whether a 17-year-old Thai boy with clinical features of TRPS and 5 supernumerary teeth had mutation in TRPS1. We report here that Trps1 is expressed during mouse tooth development, and that an individual with TRPS with supernumerary teeth has the amino acid substitution A919V in the GATA zinc finger of TRPS1. These results suggest a role for TRPS1 in tooth morphogenesis.