Type I and type V procollagen triple helix uses different subsets of the molecular ensemble for lysine posttranslational modifications in the rER.

Collagen is the most abundant protein in humans. It has a characteristic triple-helix structure and is heavily posttranslationally modified. The complex biosynthesis of collagen involves processing by many enzymes and chaperones in the rough endoplasmic reticulum. Lysyl hydroxylase 1 (LH1) is required to hydroxylate lysine for cross-linking and carbohydrate attachment within collagen triple helical sequences. Additionally, a recent study of prolyl 3-hydroxylase 3 (P3H3) demonstrated that this enzyme may be critical for LH1 activity; however, the details surrounding its involvement remain unclear. If P3H3 is an LH1 chaperone that is critical for LH1 activity, P3H3 and LH1 null mice should display a similar deficiency in lysyl hydroxylation. To test this hypothesis, we compared the amount and location of hydroxylysine in the triple helical domains of type V and I collagen from P3H3 null, LH1 null, and wild-type mice. The amount of hydroxylysine in type V collagen was reduced in P3H3 null mice, but surprisingly type V collagen from LH1 null mice contained as much hydroxylysine as type V collagen from wild-type mice. In type I collagen, our results indicate that LH1 plays a global enzymatic role in lysyl hydroxylation. P3H3 is also involved in lysyl hydroxylation, particularly at cross-link formation sites, but is not required for all lysyl hydroxylation sites. In summary, our study suggests that LH1 and P3H3 likely have two distinct mechanisms to recognize different collagen types and to distinguish cross-link formation sites from other sites in type I collagen.

Heat shock protein 47 and 65-kDa FK506-binding protein weakly but synergistically interact during collagen folding in the endoplasmic reticulum.

Collagen is the most abundant protein in the extracellular matrix in humans and is critical to the integrity and function of many musculoskeletal tissues. A molecular ensemble comprising more than 20 molecules is involved in collagen biosynthesis in the rough endoplasmic reticulum. Two proteins, heat shock protein 47 (Hsp47/SERPINH1) and 65-kDa FK506-binding protein (FKBP65/FKBP10), have been shown to play important roles in this ensemble. In humans, autosomal recessive mutations in both genes cause similar osteogenesis imperfecta phenotypes. Whereas it has been proposed that Hsp47 and FKBP65 interact in the rough endoplasmic reticulum, there is neither clear evidence for this interaction nor any data regarding their binding affinities for each other. In this study using purified endogenous proteins, we examined the interaction between Hsp47, FKBP65, and collagen and also determined their binding affinities and functions in vitro Hsp47 and FKBP65 show a direct but weak interaction, and FKBP65 prefers to interact with Hsp47 rather than type I collagen. Our results suggest that a weak interaction between Hsp47 and FKBP65 confers mutual molecular stability and also allows for a synergistic effect during collagen folding. We also propose that Hsp47 likely acts as a hub molecule during collagen folding and secretion by directing other molecules to reach their target sites on collagens. Our findings may explain why osteogenesis imperfecta-causing mutations in both genes result in similar phenotypes.

A Disintegrin and Metalloproteinase with Thrombospondin Motifs-5 (ADAMTS-5) Forms Catalytically Active Oligomers.

The metalloproteinase ADAMTS-5 (A disintegrin and metalloproteinase with thrombospondin motifs) degrades aggrecan, a proteoglycan essential for cartilage structure and function. ADAMTS-5 is the major aggrecanase in mouse cartilage, and is also likely to be the major aggrecanase in humans. ADAMTS-5 is a multidomain enzyme, but the function of the C-terminal ancillary domains is poorly understood. We show that mutant ADAMTS-5 lacking the catalytic domain, but with a full suite of ancillary domains inhibits wild type ADAMTS activity, in vitro and in vivo, in a dominant-negative manner. The data suggest that mutant ADAMTS-5 binds to wild type ADAMTS-5; thus we tested the hypothesis that ADAMTS-5 associates to form oligomers. Co-elution, competition, and in situ PLA experiments using full-length and truncated recombinant ADAMTS-5 confirmed that ADAMTS-5 molecules interact, and showed that the catalytic and disintegrin-like domains support these intermolecular interactions. Cross-linking experiments revealed that recombinant ADAMTS-5 formed large, reduction-sensitive oligomers with a nominal molecular mass of ∼ 400 kDa. The oligomers were unimolecular and proteolytically active. ADAMTS-5 truncates comprising the disintegrin and/or catalytic domains were able to competitively block full-length ADAMTS-5-mediated aggrecan cleavage, measured by production of the G1-EGE(373) neoepitope. These results show that ADAMTS-5 oligomerization is required for full aggrecanase activity, and they provide evidence that blocking oligomerization inhibits ADAMTS-5 activity. The data identify the surface provided by the catalytic and disintegrin-like domains of ADAMTS-5 as a legitimate target for the design of aggrecanase inhibitors.

Exploring the evolutionary origins of overimitation: a comparison across domesticated and non-domesticated canids.

When learning from others, human children tend to faithfully copy - or 'overimitate' - the actions of a demonstrator, even when these actions are irrelevant for solving the task at hand. We investigate whether domesticated dogs (Canis familiaris) and dingoes (Canis dingo) share this tendency to overimitate in three experiments. In Experiment 1, dogs and dingoes had the opportunity to solve a puzzle after watching an ostensive demonstrator who used both a relevant action and an irrelevant action. We find clear evidence against overimitation in both species. In contrast to human children (Horner & Whiten, 2005), dogs and dingoes used the irrelevant action less often across trials, suggesting that both species were filtering out the irrelevant action as they gained experience with the puzzle (like chimpanzees; Horner & Whiten, 2005). Experiments 2 and 3 provide further evidence against overimitation, demonstrating that both species' behavior is better characterized by individual exploration than overimitation. Given that both species, particularly dogs, show human-like social learning in other contexts, these findings provide additional evidence that overimitation may be a unique aspect of human social learning. A video abstract of this article can be viewed at: https://youtu.be/g2mRniJZ7aU.

Assessing the susceptibility of transgenic mice overexpressing deer prion protein to bovine spongiform encephalopathy.

Several transgenic mouse models have been developed which facilitate the transmission of chronic wasting disease (CWD) of cervids and allow prion strain discrimination. The present study was designed to assess the susceptibility of the prototypic mouse line, Tg(CerPrP)1536(+/-), to bovine spongiform encephalopathy (BSE) prions, which have the ability to overcome species barriers. Tg(CerPrP)1536(+/-) mice challenged with red deer-adapted BSE resulted in 90% to 100% attack rates, and BSE from cattle failed to transmit, indicating agent adaptation in the deer.

The expanded collagen VI family: new chains and new questions.

Collagen VI is a component of the extracellular matrix of almost all connective tissues, including cartilage, bone, tendon, muscles and cornea, where it forms abundant and structurally unique microfibrils organized into different suprastructural assemblies. The precise role of collagen VI is not clearly defined although it is most abundant in the interstitial matrix of tissues and often found in close association with basement membranes. Three genetically distinct collagen VI chains, α1(VI), α2(VI) and α3(VI), encoded by the COL6A1. COL6A2 and COL6A3 genes, were first described more than 20 years ago. Their molecular assembly and role in congenital muscular dystrophy has been broadly characterized. In 2008, three additional collagen VI genes arrayed in tandem at a single gene locus on chromosome 3q in humans, and chromosome 9 in mice, were described. Following the naming scheme for collagens the new genes were designated COL6A4. COL6A5 and COL6A6 encoding the α4(VI), α5(VI) and α6(VI) chains, respectively. This review will focus on the current state of knowledge of the three new chains.

A novel form of chondrocyte stress is triggered by a COMP mutation causing pseudoachondroplasia.

Pseudoachondroplasia (PSACH) results from mutations in cartilage oligomeric matrix protein (COMP) and the p.D469del mutation within the type III repeats of COMP accounts for approximately 30% of PSACH. To determine disease mechanisms of PSACH in vivo, we introduced the Comp D469del mutation into the mouse genome. Mutant animals were normal at birth but grew slower than their wild-type littermates and developed short-limb dwarfism. In the growth plates of mutant mice chondrocyte columns were reduced in number and poorly organized, while mutant COMP was retained within the endoplasmic reticulum (ER) of cells. Chondrocyte proliferation was reduced and apoptosis was both increased and spatially dysregulated. Previous studies on COMP mutations have shown mutant COMP is co-localized with chaperone proteins, and we have reported an unfolded protein response (UPR) in mouse models of PSACH-MED (multiple epiphyseal dysplasia) harboring mutations in Comp (T585M) and Matn3, Comp etc (V194D). However, we found no evidence of UPR in this mouse model of PSACH. In contrast, microarray analysis identified expression changes in groups of genes implicated in oxidative stress, cell cycle regulation, and apoptosis, which is consistent with the chondrocyte pathology. Overall, these data suggest that a novel form of chondrocyte stress triggered by the expression of mutant COMP is central to the pathogenesis of PSACH.

Fibroblast growth factor receptor 3 (FGFR3) is a strong heat shock protein 90 (Hsp90) client: implications for therapeutic manipulation.

Fibroblast growth factor receptor 3 (FGFR3) is a key regulator of growth and differentiation, whose aberrant activation causes a number of genetic diseases including achondroplasia and cancer. Hsp90 is a specialized molecular chaperone involved in stabilizing a select set of proteins termed clients. Here, we delineate the relationship of Hsp90 and co-chaperone Cdc37 with FGFR3 and the FGFR family. FGFR3 strongly associates with these chaperone complexes and depends on them for stability and function. Inhibition of Hsp90 function using the geldanamycin analog 17-AAG induces the ubiquitination and degradation of FGFR3 and reduces the signaling capacity of FGFR3. Other FGFRs weakly interact with these chaperones and are differentially influenced by Hsp90 inhibition. The Hsp90-related ubiquitin ligase CHIP is able to interact and destabilize FGFR3. Our results establish FGFR3 as a strong Hsp90 client and suggest that modulating Hsp90 chaperone complexes may beneficially influence the stability and function of FGFR3 in disease.

Understanding Racial, Ethnic, and Socioeconomic Differences in the Ambulatory Care Experience.

BACKGROUND AND OBJECTIVE: Racial and ethnic and socioeconomic differences in patient experience are prevalent and can negatively impact health outcomes. Our objective was to examine differences in family experience of care in the pediatric ambulatory setting. METHODS: We conducted interviews with parents of patients from different demographic groups who had received care at 1 of 3 clinics at a quaternary children's hospital. Multidisciplinary team conducted inductive and deductive thematic analysis of transcribed interviews. Sentiments and recurring themes were compared within and between racial and ethnic groups, insurance status, and language. RESULTS: Eighty parents were interviewed. Three primary themes were identified: (1) mitigation of system issues: parents' mixed experiences with staff or clinicians mitigating system issues impacted their overall perceptions of care; (2) pivotal role of personal interactions: clinicians' interactions positively influenced family-clinician relationships and offset negative experiences; (3) effective explanations: clinicians' clear and thorough explanations were crucial in enhancing parent confidence in care. As an overarching theme, discrimination and disrespect by staff undermined trust in care, affecting all aspects of experience. With the exception of explanations, a higher proportion of publicly-insured parents reported negative experiences across all themes compared to those with private insurance. Asian parents with public insurance had the highest proportion of interviews that were mainly negative in sentiment. CONCLUSIONS: Our findings offer nuanced insights into differences in the experience of ambulatory care. Insurance status emerged as an important marker of differential perceptions of care. Our study points to areas for improvement and highlights family-clinician interactions as vital to overall positive experience.

Cartilage oligomeric matrix protein promotes cell attachment via two independent mechanisms involving CD47 and alphaVbeta3 integrin.

Cartilage oligomeric matrix protein (COMP) is a pentameric approximately 524 kDa multidomain extracellular matrix protein and is the fifth member of the thrombospondin family. COMP is abundantly expressed in proliferating and hypertrophic chondrocytes of the growth plate, articular cartilage, synovium, tendon, and ligament. The spatial localization of COMP highlights its importance in the phenotypes of pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED), COMP disorders that are characterized by disproportionate short stature, brachydactyly, scoliosis, early-onset osteoarthritis, and joint hypermobility. In this study, the role of COMP in ligament was investigated with a series of cell attachment assays using ligament cells binding to COMP. A dose-dependent cell attachment activity was found, which was inhibited by a peptide containing the SFYVVMWK amino acid sequence derived from the globular C-terminal domain of COMP. This activity was independent of the recently described RGD-dependent attachment activity. Function-blocking antibodies to CD47 and alphaVbeta3 integrin reduced cell attachment to COMP, implicating the participation of these cell surface molecules in COMP cell binding. Immunofluorescence studies showed that cell attachment to COMP induced the formation of lamellae containing F-actin microspikes associated with fascin. We propose that COMP promotes cell attachment via two independent mechanisms involving cell surface CD47 and alphaVbeta3 integrin and that a consequence of cell attachment to COMP is the specific induction of fascin-stabilized actin microspikes.

Lemierre syndrome: a pediatric case series and review of literature.

BACKGROUND: Lemierre syndrome is a rare disease of the head and neck often affecting adolescents and young adults. Classically, infection begins in the oropharynx with thrombosis of the tonsillar veins followed by involvement of the parapharyngeal space and the internal jugular vein. Septicemia and pulmonary lesions develop as infection spreads via septic emboli. Although a rare entity in modern times, Lemierre syndrome remains a disease of considerable morbidity and potential mortality. METHODS: This was a retrospective review of 3 cases and associated literature. RESULTS: A common 1- to 2-week history of fever, sore throat, neck pain, and fatigue was observed in all patients. Patient 1 developed right facial swelling, neck tenderness, trismus, and tonsillar exudate. Patient 2 displayed right tonsillar erythema and enlargement with right neck tenderness. Patient 3 revealed bilateral tonsillar enlargement with exudate and left neck tenderness. Subsequent studies included blood cultures and computed tomography, after which empiric antibiotic therapy was started. Patient 1 underwent drainage of a right peritonsillar abscess, right pressure equalization tube placement, and ligation of the right external jugular vein. He subsequently developed subdural empyemas, cavernous sinus thrombosis, and carotid artery narrowing and required 9 weeks of antibiotic therapy. Patients 2 and 3 developed pulmonary lesions and received 6 weeks of antibiotic therapy. Timing was crucial in all cases. CONCLUSIONS: Lemierre syndrome is a rare but severe opportunistic infection with poor prognostic outcomes if left untreated. Early diagnosis and treatment is essential. Aggressive antibiotic therapy coupled with surgical intervention, when necessary, provides excellent outcomes.

The novel missense mutation Met48Lys in FKBP22 changes its structure and functions.

Mutations in the FKBP14 gene encoding FKBP22 (FK506 Binding Protein 22 kDa) cause kyphoscoliotic Ehlers-Danlos Syndrome (kEDS). The first clinical report showed that a lack of FKBP22 protein due to mutations causing nonsense-mediated decay of the mRNA leads to a wide spectrum of clinical phenotypes including progressive kyphoscoliosis, joint hypermobility, hypotonia, hyperelastic skin, hearing loss and aortic rupture. Our previous work showed that these phenotypic features could be correlated with the functions of FKBP22, which preferentially binds to type III, VI and X collagens, but not to type I, II or V collagens. We also showed that FKBP22 catalyzed the folding of type III collagen through its prolyl isomerase activity and acted as a molecular chaperone for type III collagen. Recently, a novel missense mutation Met48Lys in FKBP22 was identified in a patient with kEDS. In this report, we expand the list of substrates of FKBP22 and also demonstrate that the Met48Lys mutation diminishes the activities of FKBP22, indicating that pathology can arise from absence of FKBP22, or partial loss of its function.

Sprouty 2 disturbs FGFR3 degradation in thanatophoric dysplasia type II: a severe form of human achondroplasia.

Thanatophoric dysplasia is a member of the achondroplasia family of human skeletal dysplasias, which result from FGFR3 mutations that exaggerate this receptor's inhibitory influence on chondrocyte proliferation and differentiation in the skeletal growth plate. We have previously reported that defective lysosomal degradation of activated receptor contributes to the gain-of-function of the mutant FGFR3. We now provide evidence that this disturbance is mediated by the receptor's kinase activity and involves constitutive induction and activation of Spry2. Our findings suggest that activated Spry2 may interfere with c-Cbl-mediated ubiquitination of FGFR3 by sequestering c-Cbl. They provide novel insight into the pathogenesis of this group of human skeletal dysplasias and identify a mechanism that potentially could be targeted therapeutically.

The effects of laser irradiation of cartilage on chondrocyte gene expression and the collagen matrix.

OBJECTIVES: Laser reshaping of cartilage is an emerging technology aimed at replacing conventional techniques for aesthetic and reconstructive surgery. Little is known about the mechanisms of wound healing following the photothermal heating during laser reshaping and, ultimately, how collagen remodels in the irradiated tissue. Healthy hyaline and elastic cartilage as found in the ear, nose, larynx, and trachea does not express collagen type I which is characteristic of fibro-cartilage and scar tissue. The aim of the study was to determine if collagen I and II gene expression occurs within laser irradiated rabbit septal cartilage. METHODS: Nasal septum harvested from freshly euthanized New Zealand White rabbits were irradiated with an Nd:YAG laser. After 2 weeks in culture, the laser spot and surrounding non-irradiated regions were imaged using immunofluorescence staining and evaluated using reverse transcription polymerase chain reaction (RT-PCR) to determine the presence of collagen I and II, and ascertain collagen I and II gene expression, respectively. RESULTS: All laser irradiated specimens showed a cessation in collagen II gene expression within the center of the laser spot. Collagen II was expressed in the surrounding region encircling the laser spot and within the non-irradiated periphery in all specimens. Immunohistochemistry identified only type II collagen. Neither collagen I gene expression nor immunoreactivity were identified in any specimens regardless or irradiation parameters. CONCLUSIONS: Laser irradiation of rabbit septal cartilage using dosimetry parameters similar to those used in laser reshaping does not result in the detection of either collagen I gene expression or immunoreactivity. Only collagen type II was noted after laser exposure in vitro following cell culture, which suggests that the cellular response to laser irradiation is distinct from that observed in conventional wound healing. Laser irradiation of cartilage can leave an intact collagen matrix which likely allows chondrocyte recovery on an intact scaffold.

Thermoforming of tracheal cartilage: viability, shape change, and mechanical behavior.

BACKGROUND AND OBJECTIVES: Trauma, emergent tracheostomy, and prolonged intubation are common causes of severe deformation and narrowing of the trachea. Laser technology may be used to reshape tracheal cartilage using minimally invasive methods. The objectives of this study were to determine: (1) the dependence of tracheal cartilage shape change on temperature and laser dosimetry using heated saline bath immersion and laser irradiation, respectively, (2) the effect of temperature on the mechanical behavior of cartilage, and (3) tissue viability as a function of laser dosimetry. MATERIALS AND METHODS: Ex vivo rabbit trachea cartilage specimens were bent and secured around a cylinder (6 mm), and then immersed in a saline bath (45 and 72 degrees C) for 5-100 seconds. In separate experiments, tracheal specimens were irradiated with a diode laser (lambda = 1.45 microm, 220-400 J/cm(2)). Mechanical analysis was then used to determine the elastic modulus in tension after irradiation. Fluorescent viability assays combined with laser scanning confocal microscopy (LSCM) were employed to image and identify thermal injury regions. RESULTS: Shape change transition zones, between 62 and 66 degrees C in the saline heating bath and above power densities of 350 J/cm(2) (peak temperatures 65+/-10 degrees C) for laser irradiation were identified. Above these zones, the elastic moduli were higher (8.2+/-4 MPa) than at lower temperatures (4.5+/-3 MPa). LSCM identified significant loss of viable chondrocytes within the laser-irradiation zones. CONCLUSION: Our results indicate a change in mechanical properties occurs with laser irradiation and further demonstrates that significant thermal damage is concurrent with clinically relevant shape change in the elastic cartilage tissues of the rabbit trachea using the present laser and dosimetry parameters.

Long-term botulinum toxin dose consistency for treatment of adductor spasmodic dysphonia.

OBJECTIVES: Botulinum toxin (BTX) injection is currently the primary and most common treatment for adductor spasmodic dysphonia (ADSD). A variety of injection strategies and dosage regimens have been described. This study reports on our experience with the dosage schedule and dosing consistency of BTX for the treatment of ADSD. METHODS: We retrospectively reviewed our laryngeal BTX database for the period 1991 to 2005. Our strict inclusion requirements limited our selection to 13 patients who had received a minimum of 6 injections (average, 11.5; range, 6 to 19) of BTX for ADSD. RESULTS: The average total dose of BTX to the larynx for each treatment episode was 3.9 units (range, 1.5 to 7.5). The total dose administered tended to trend downward among patients who began treatment from 1991 to 1998, indicating that the initial dose (usually 2.5 units per side) may have been high. Those patients who began from 1999 onward had a more stable dose, indicating that the initial dose (usually 1.5 units per side) was more suitable. The subjects underwent an average of 2.2 injections (range, 1 to 5) before reaching their optimal BTX dose. The total number of treatments performed in this group of patients was 150, of which 145 were successful (96.7%). CONCLUSIONS: The BTX dose for the optimal treatment of ADSD usually remains consistent over time, as does the treatment interval. An initial dose of 1.5 units per side or less appears to improve dosing stability, indicating that the initial dosing of 2.5 units per side in our study was often greater than required. The optimal BTX dose was usually ascertained by the second or third injection. In our patient population, the long-term dosing consistency of BTX confirmed that neither tachyphylaxis nor increasing sensitivity to BTX occurred during the course of treatment for ADSD.

Authentication of collagen VI antibodies.

BACKGROUND: Collagen VI is a ubiquitously-expressed macromolecule that forms unique microfibrillar assemblies in the extracellular matrix. Mutations in the COL6A1, COL6A2 and COL6A3 genes result in congenital muscular dystrophy, arguing that collagen is critical for skeletal muscle development and function. Antibodies against collagen VI are important clinical and diagnostic tools in muscular dystrophy. They are used to confirm genetic findings by detecting abnormalities in the distribution, organization and overall levels of collagen VI in cells and tissues isolated from patients. METHODS: Many antibodies have been raised against tissue-purified collagen VI and individual collagen VI chains, however few have been properly validated for sensitivity and chain specificity. To address this deficiency, we compared the ability of 23 commercially-available antibodies to detect extracellular collagen VI by immunohistochemistry on frozen tissue sections. To determine chain specificity, immunoblot analyses were conducted on cell lysates isolated from cells transfected with cDNAs for each individual chain and cells expressing all three chains together. RESULTS: Our analyses identified 15 antibodies that recognized tissue collagen VI by immunohistochemistry at varying intensities and 20 that successfully detected collagen VI by immunoblotting. Three antibodies failed to recognize collagen VI by either method under the conditions tested. All chain-specific antibodies that worked by immunoblotting specifically recognized their correct chain, and no other chains. CONCLUSIONS: This series of side-by-side comparisons reveal at least two antibodies specific for each chain that work well for immunohistochemistry on frozen sections. This validation study expands the repertoire of antibodies available for muscular dystrophy studies caused by defects in collagen VI.

Novel compound heterozygous mutations in inositol polyphosphate phosphatase-like 1 in a family with severe opsismodysplasia.

This study aimed to identify the genetic basis of a severe skeletal lethal dysplasia. The main clinical features of two affected fetuses included short limbs with flared metaphyses, bowed radii, femora and tibiae, irregular ossification of hands and feet, and marked platyspondyly. Affected and nonaffected family members were subjected to whole-exome sequencing, followed by immunoblot analysis on amniocytes isolated from one of the affected individuals. Unique compound heterozygous variants in the inositol polyphosphate phosphatase-like 1 (INPPL1) gene encoding the SHIP2 protein were identified in both affected individuals. One variant was inherited from each unaffected parent. Both allelic variants, c.(2327-1G>C);(1150_1151delGA), are predicted to result in premature stop codons leading to nonsense-mediated mRNA decay of the mutant alleles and no production of SHIP2. The absence of SHIP2 was confirmed by immunoblot analysis of proband amniocytes. This skeletal disorder is caused by the complete absence of the SHIP2 protein. INPPL1 mutations have been reported in opsismodysplasia, an autosomal recessive skeletal dysplasias with significant delayed bone formation. Our finding highlights the critical role that INPPL1/SHIP2 plays in skeletal development.

Hypoxia-inducible factor 3-alpha expression is associated with the stable chondrocyte phenotype.

The hypoxia-inducible factors HIF-1α and HIF-2α are important regulators of the chondrocyte phenotype but little is known about HIF-3α in cartilage. The objective of this study was to characterize HIF-3α (HIF3A) expression during chondrocyte differentiation in vitro and in native cartilage tissues. HIF3A, COL10A1, and MMP13 were quantified in mesenchymal stem cells (MSCs) and articular chondrocytes from healthy and osteoarthritic (OA) tissue in three-dimensional cultures and in human embryonic epiphyses and adult articular cartilage. HIF3A was found to have an inverse association with hypertrophic markers COL10A1 and MMP13 in chondrogenic cells and tissues. In healthy chondrocytes, HIF3A was induced by dexamethasone and increased during redifferentiation. By comparison, HIF3A expression was extremely low in chondrogenically differentiated MSCs expressing high levels of COL10A1 and MMP13. HIF3A was also lower in redifferentiated OA chondrocytes than in healthy chondrocytes. In human embryonic epiphyseal tissue, HIF3A expression was lowest in the hypertrophic zone. Distinct splice patterns were also found in embryonic cartilage when compared with adult articular cartilage and redifferentiated chondrocytes. These in vitro and in vivo findings suggest that HIF3A levels are indicative of the hypertrophic state of chondrogenic cells and one or more splice variants may be important regulators of the chondrocyte phenotype.

PHENOTYPIC VARIABILITY IN INDIVIDUALS WITH TYPE V OSTEOGENESIS IMPERFECTA WITH IDENTICAL IFITM5 MUTATIONS.

BACKGROUND: Osteogenesis imperfecta (OI) type V is a dominantly inherited skeletal dysplasia characterized by fractures and progressive deformity of long bones. In addition, patients often present with radial head dislocation, hyperplastic callus, and calcification of the forearm interosseous membrane. Recently, a specific mutation in the IFITM5 gene was found to be responsible for OI type V. This mutation, a C to T transition 14 nucleotides upstream from the endogenous start codon, creates a new start methionine that appears to be preferentially used by the translational machinery. However, the mechanism by which the lengthened protein results in a dominant type of OI is unknown. METHODS AND RESULTS: We report 7 ethnically diverse (African-American, Caucasian, Hispanic, and African) individuals with OI type V from 2 families and 2 sporadic cases. Exome sequencing failed to identify a causative mutation. Using Sanger sequencing, we found that all affected individuals in our cohort possess the c.-14 IFITM5 variant, further supporting the notion that OI type V is caused by a single, discrete mutation. Our patient cohort demonstrated inter-and intrafamilial phenotypic variability, including a father with classic OI type V whose daughter had a phenotype similar to OI type I. This clinical variability suggests that modifier genes influence the OI type V phenotype. We also confirm that the mutation creates an aberrant IFITM5 protein containing an additional 5 amino acids at the N-terminus. CONCLUSIONS: The variable clinical signs in these cases illustrate the significant variability of the OI type V phenotype caused by the c.-14 IFITM5 mutation. The affected individuals are more ethnically diverse than previously reported.