Dobrawa Napierala

Mutations in SERPINF1 cause osteogenesis imperfecta type VI

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 (SERPINF1) 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.

Mutations in FKBP10 cause recessive osteogenesis imperfecta and bruck syndrome

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.

Uncoupling of chondrocyte differentiation and perichondrial mineralization underlies the skeletal dysplasia in tricho-rhino-phalangeal syndrome

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.

Runx2 contributes to murine Col10a1 gene regulation through direct interaction with its cis-enhancer

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.

Tricho-Rhino-Phalangeal Syndrome with Supernumerary Teeth:

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.

Differential Effects of Collagen Prolyl 3-Hydroxylation on Skeletal Tissues

Mutations in the genes encoding cartilage associated protein (CRTAP) and prolyl 3-hydroxylase 1 (P3H1 encoded by LEPRE1) were the first identified causes of recessive Osteogenesis Imperfecta (OI). These proteins, together with cyclophilin B (encoded by PPIB), form a complex that 3-hydroxylates a single proline residue on the α1(I) chain (Pro986) and has cis/trans isomerase (PPIase) activity essential for proper collagen folding. Recent data suggest that prolyl 3-hydroxylation of Pro986 is not required for the structural stability of collagen; however, the absence of this post-translational modification may disrupt protein-protein interactions integral for proper collagen folding and lead to collagen over-modification. P3H1 and CRTAP stabilize each other and absence of one results in degradation of the other. Hence, hypomorphic or loss of function mutations of either gene cause loss of the whole complex and its associated functions. The relative contribution of losing this complexs 3-hydroxylation versus PPIase and collagen chaperone activities to the phenotype of recessive OI is unknown. To distinguish between these functions, we generated knock-in mice carrying a single amino acid substitution in the catalytic site of P3h1 (Lepre1H662A). This substitution abolished P3h1 activity but retained ability to form a complex with Crtap and thus the collagen chaperone function. Knock-in mice showed absence of prolyl 3-hydroxylation at Pro986 of the α1(I) and α1(II) collagen chains but no significant over-modification at other collagen residues. They were normal in appearance, had no growth defects and normal cartilage growth plate histology but showed decreased trabecular bone mass. This new mouse model recapitulates elements of the bone phenotype of OI but not the cartilage and growth phenotypes caused by loss of the prolyl 3-hydroxylation complex. Our observations suggest differential tissue consequences due to selective inactivation of P3H1 hydroxylase activity versus complete ablation of the prolyl 3-hydroxylation complex.

Localization of the cis-enhancer element for mouse type X collagen expression in hypertrophic chondrocytes in vivo.

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 (−4.4 to −3.8 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 (−4296 to −4147 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 5′‐ and 3′‐ends (−4276 to −4243 and −4166 to −4152 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 tissue‐specific 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.

Characterization of a New Syndrome That Associates Craniosynostosis, Delayed Fontanel Closure, Parietal Foramina, Imperforate Anus, and Skin Eruption: CDAGS

We describe the clinical characterization, molecular analyses, and genetic mapping of a distinct genetic condition characterized by craniosynostosis, delayed closure of the fontanel, cranial defects, clavicular hypoplasia, anal and genitourinary malformations, and skin eruption. We have identified seven patients with this phenotype in four families from different geographic regions and ethnic backgrounds. This is an autosomal recessive condition that brings together apparently opposing pathophysiologic and developmental processes, including accelerated suture closure and delayed ossification. Selected candidate genes--including RUNX2, CBFB, MSX2, ALX4, TWIST1, and RECQL4--were screened for mutations, by direct sequencing of their coding regions, and for microdeletions, by fluorescent in situ hybridization. No mutations or microdeletions were detected in any of the genes analyzed. A genomewide screen yielded the maximum estimated LOD score of +2.38 for markers D22S283 and D22S274 on chromosome 22q12-q13. We hypothesize that the gene defect in this condition causes novel context-dependent dysregulation of multiple signaling pathways, including RUNX2, during osteoblast differentiation and craniofacial morphogenesis.

Transcriptional repression of the Dspp gene leads to dentinogenesis imperfecta phenotype in Col1a1-Trps1 transgenic mice

Dentinogenesis imperfecta (DGI) is a hereditary defect of dentin, a calcified tissue that is the most abundant component of teeth. Most commonly, DGI is manifested as a part of osteogenesis imperfecta (OI) or the phenotype is restricted to dental findings only. In the latter case, DGI is caused by mutations in the DSPP gene, which codes for dentin sialoprotein (DSP) and dentin phosphoprotein (DPP). Although these two proteins together constitute the majority of noncollagenous proteins of the dentin, little is known about their transcriptional regulation. Here we demonstrate that mice overexpressing the Trps1 transcription factor (Col1a1‐Trps1 mice) in dentin‐producing cells, odontoblasts, present with severe defects of dentin formation that resemble DGI. Combined micro–computed tomography (µCT) and histological analyses revealed tooth fragility due to severe hypomineralization of dentin and a diminished dentin layer with irregular mineralization in Col1a1‐Trps1 mice. Biochemical analyses of noncollagenous dentin matrix proteins demonstrated decreased levels of both DSP and DPP proteins in Col1a1‐Trps1 mice. On the molecular level, we demonstrated that sustained high levels of Trps1 in odontoblasts lead to dramatic decrease of Dspp expression as a result of direct inhibition of the Dspp promoter by Trps1. During tooth development Trps1 is highly expressed in preodontoblasts, but in mature odontoblasts secreting matrix its expression significantly decreases, which suggests a Trps1 role in odontoblast development. In these studies we identified Trps1 as a potent inhibitor of Dspp expression and the subsequent mineralization of dentin. Thus, we provide novel insights into mechanisms of transcriptional dysregulation that leads to DGI.

Dual Role of the Trps1 Transcription Factor in Dentin Mineralization

Background: Regulation of dentin mineralization at the gene expression level is poorly understood. Results: Trps1 supports expression of osteogenic genes Alpl, Phospho1, Runx2, and Sp7 in preodontoblastic cells, and in mature cells Trps1 represses phosphate metabolism genes Phex and Vdr. Conclusion: The role of Trps1 in mineralization depends on odontoblastic differentiation stage. Significance: These findings provide insights into regulation of odontoblastic maturation and function. TRPS1 (tricho-rhino-phalangeal syndrome) is a unique GATA-type transcription factor that acts as a transcriptional repressor. TRPS1 deficiency and dysregulated TRPS1 expression result in skeletal and dental abnormalities implicating TRPS1 in endochondral bone formation and tooth development. Moreover, patients with tricho-rhino-phalangeal syndrome frequently present with low bone mass indicating TRPS1 involvement in bone homeostasis. In addition, our previous data demonstrated accelerated mineralization of the perichondrium in Trps1 mutant mice and impaired dentin mineralization in Col1a1-Trps1 transgenic mice, implicating Trps1 in the mineralization process. To understand the role of Trps1 in the differentiation and function of cells producing mineralized matrix, we used a preodontoblastic cell line as a model of dentin mineralization. We generated both Trps1-deficient and Trps1-overexpressing stable cell lines and analyzed the progression of mineralization by alkaline phosphatase and alizarin red staining. As predicted, based on our previous in vivo data, delayed and decreased mineralization of Trps1-overexpressing odontoblastic cells was observed when compared with control cells. This was associated with down-regulation of genes regulating phosphate homeostasis. Interestingly, Trps1-deficient cells lost the ability to mineralize and demonstrated decreased expression of several genes critical for initiating the mineralization process, including Alpl and Phospho1. Based on these data, we have concluded that Trps1 serves two critical and context-dependent functions in odontoblast-regulated mineralization as follows: 1) Trps1 is required for odontoblast maturation by supporting expression of genes crucial for initiating the mineralization process, and 2) Trps1 represses the function of mature cells and, consequently, restricts the extent of extracellular matrix mineralization.