Signature of circulating small non-coding RNAs during early fracture healing in mice.

Fracture healing is a complex process with multiple overlapping metabolic and differentiation phases. Small non-coding RNAs are involved in the regulation of fracture healing and their presence in circulation is under current interest due to their obvious value as potential biomarkers. Circulating microRNAs (miRNAs) have been characterized to some extent but the current knowledge on tRNA-derived small RNA fragments (tsRNAs) is relatively scarce, especially in circulation. In this study, the spectrum of circulating miRNAs and tsRNAs was analysed by next generation sequencing to show their differential expression during fracture healing in vivo. Analysed tsRNA fragments included stress-induced translation interfering tRNA fragments (tiRNAs or tRNA halves) and internal tRNA fragments (i-tRF), within the size range of 28-36 bp. To unveil the expression of these non-coding RNAs, genome-wide analysis was performed on two months old C57BL/6 mice on days 1, 5, 7, 10, and 14 (D1, D5, D7, D10, and D14) after a closed tibial fracture. Valine isoacceptor tRNA-derived Val-AAC 5'end and Val-CAC 5'end fragments were the major types of 5'end tiRNAs in circulation, comprising about 65 % of the total counts. Their expression was not affected by fracture. After a fracture, the levels of two 5'end tiRNAs Lys-TTT 5' and Lys-CTT 5' were decreased and His-GTG 5' was increased through D1-D14. The level of miR-451a was decreased on the first post-fracture day (D1), whereas miR-328-3p, miR-133a-3p, miR-375-3p, miR-423-5p, and miR-150-5p were increased post-fracture. These data provide evidence on how fracture healing could provoke systemic metabolic effects and further pinpoint the potential of small non-coding RNAs as biomarkers for tissue regeneration.

Mesenchymal cell-derived Wnt1 signaling regulates subchondral bone remodeling but has no effects on the development of growth plate or articular cartilage in mice.

Chondrocyte differentiation is a principal progress in endochondral ossification and in the formation of secondary ossification center (SOC) during the long bone development. We have previously reported that targeted deletion of Wnt1 in mesenchymal progenitors (Wnt1Prrx-/-) leads to spontaneous fractures and severe osteopenia in mouse long bones, suggesting that Wnt1 is a key regulator of bone metabolism. However, the effect of Wnt1 on the regulation of cartilage development and chondrocyte differentiation remained unknown. In this study, WNT1 protein expression was observed in lateral superficial cartilage and growth plate pre-hypertrophic chondrocytes in mice. Wnt1 mRNA expression was detected in epiphyseal cartilage from E16.5 to 3 month-old mice. Detailed histological analyses revealed that the average thickness and chondrocyte density of proximal tibial articular cartilage and growth plate were unchanged between Wnt1Prrx-/- and control mice. However, µCT analysis of tibial epiphyses showed that the subchondral bone mass was reduced in Wnt1Prrx-/- mice compared to control mice, as demonstrated by decreased bone volume, trabecular number, trabecular thickness, and increased trabecular separation in Wnt1Prrx-/- mice. Mechanistically, histomorphometric analyses showed that the reduced subchondral bone mass in Wnt1Prrx-/- mice was due to impaired bone formation and enhanced bone resorption. In vitro, exogenous Wnt1 inhibited chondrogenesis and chondrocyte hypertrophy in both cell autonomous and juxtacrine manners, while matrix mineralization and the expression of Mmp13, Mmp9 and Opn were induced in a juxtacrine manner. Taken together, mesenchymal cell-derived Wnt1 is an important regulator of subchondral bone remodeling, although it has no effect on the regulation of growth plate or articular cartilage.

Multiple targets identified with genome wide profiling of small RNA and mRNA expression are linked to fracture healing in mice.

Long-bone fracture is a common injury and its healing process at the fracture site involves several overlapping phases, including inflammation, migration of mesenchymal progenitors into the fracture site, endochondral ossification, angiogenesis and finally bone remodelling. Increasing evidence shows that small noncoding RNAs are important regulators of chondrogenesis, osteogenesis and fracture healing. MicroRNAs are small single-stranded, non-coding RNA-molecules intervening in most physiological and biological processes, including fracture healing. Angiogenin-cleaved 5' tRNA halves, also called as tiRNAs (stress-induced RNAs) have been shown to repress protein translation. In order to gain further understanding on the role of small noncoding RNAs in fracture healing, genome wide expression profiles of tiRNAs, miRNAs and mRNAs were followed up to 14 days after fracture in callus tissue of an in vivo mouse model with closed tibial fracture and, compared to intact bone and articular cartilage at 2 months of age. Total tiRNA expression level in cartilage was only approximately one third of that observed in control D0 bone. In callus tissue, 11 mature 5'end tiRNAs out of 191 tiRNAs were highly expressed, and seven of them were differentially expressed during fracture healing. When comparing the control tissues, 25 miRNAs characteristic to bone and 29 miRNAs characteristic to cartilage tissue homeostasis were identified. Further, a total of 54 out of 806 miRNAs and 5420 out of 18,700 mRNAs were differentially expressed (DE) in callus tissue during fracture healing and, in comparison to control bone. They were associated to gene ontology processes related to mesenchymal tissue development and differentiation. A total of 581 miRNA-mRNA interactions were identified for these 54 DE miRNAs by literature searches in PubMed, thereby linking by Spearman correlation analysis 14 downregulated and 28 upregulated miRNAs to 164 negatively correlating and 168 positively correlating miRNA-mRNA pairs with chondrogenic and osteogenic phases of fracture healing. These data indicated that tiRNAs and miRNAs were differentially expressed in fracture callus tissue, suggesting them important physiological functions during fracture healing. Hence, the data provided by this study may contribute to future clinical applications, such as potential use as biomarkers or as tools in the development of novel therapeutic approaches for fracture healing.

Soluble activin type IIB receptor improves fracture healing in a closed tibial fracture mouse model.

Fractures still present a significant burden to patients due to pain and periods of unproductivity. Numerous growth factors have been identified to regulate bone remodeling. However, to date, only the bone morphogenetic proteins (BMPs) are used to enhance fracture healing in clinical settings. Activins are pleiotropic growth factors belonging to the TGF-ß superfamily. We and others have recently shown that treatment with recombinant fusion proteins of activin receptors greatly increases bone mass in different animal models by trapping activins and other ligands thus inhibiting their signaling pathways. However, their effects on fracture healing are less known. Twelve-week old male C57Bl mice were subjected to a standardized, closed tibial fracture model. Animals were divided into control and treatment groups and were administered either PBS control or a soluble activin type IIB receptor (ActRIIB-Fc) intraperitoneally once a week for a duration of two or four weeks. There were no significant differences between the groups at two weeks but we observed a significant increase in callus mineralization in ActRIIB-Fc-treated animals by microcomputed tomography imaging at four weeks. Bone volume per tissue volume was 60%, trabecular number 55% and bone mineral density 60% higher in the 4-week calluses of the ActRIIB-Fc-treated mice (p<0.05 in all). Biomechanical strength of 4-week calluses was also significantly improved by ActRIIB-Fc treatment as stiffness increased by 64% and maximum force by 45% (p<0.05) compared to the PBS-injected controls. These results demonstrate that ActRIIB-Fc treatment significantly improves healing of closed long bone fractures. Our findings support the previous reports of activin receptors increasing bone mass but also demonstrate a novel approach for using ActRIIB-Fc to enhance fracture healing.

Aging and serum exomiR content in women-effects of estrogenic hormone replacement therapy.

Exosomes participate in intercellular messaging by transporting bioactive lipid-, protein- and RNA-molecules and -complexes. The contents of the exosomes reflect the physiological status of an individual making exosomes promising targets for biomarker analyses. In the present study we extracted exosome microRNAs (exomiRs) from serum samples of premenopausal women (n = 8) and monozygotic postmenopausal twins (n = 10 female pairs), discordant for the use of estrogenic hormone replacement therapy (HRT), in order to see whether the age or/and the use of HRT associates with exomiR content. A total of 241 exomiRs were detected by next generation sequencing, 10 showing age, 14 HRT and 10 age +HRT -related differences. When comparing the groups, differentially expressed miRs were predicted to affect cell proliferation processes showing inactivation with younger age and HRT usage. MiR-106-5p, -148a-3p, -27-3p, -126-5p, -28-3p and -30a-5p were significantly associated with serum 17ß-estradiol. MiRs formed two hierarchical clusters being indicative of positive or negative health outcomes involving associations with body composition, serum 17ß-estradiol, fat-, glucose- and inflammatory markers. Circulating exomiR clusters, obtained by NGS, could be used as indicators of metabolic and inflammatory status affected by hormonal changes at menopause. Furthermore, the individual effects of HRT-usage could be evaluated based on the serum exomiR signature.

Reduced expression of Sfrp1 during chondrogenesis and in articular chondrocytes correlates with osteoarthritis in STR/ort mice.

To circumvent the problems of genetic and environmental diversity hampering the analysis in humans, we turned to a murine model for human knee osteoarthritis (OA) and fine mapped a previously defined OA-quantitative trait locus (QTL). We here focused on one of the candidate genes within the OA-QTL encoding the Wnt antagonist secreted frizzled related protein 1 (Sfrp1). Sequence analysis of the Sfrp1 gene in the OA strain STR/ort revealed 23 polymorphic changes with a potential to alter the gene expression. Indeed, a reduced expression in STR/ort mice was demonstrated for articular chondrocytes and hypertrophic chondrocytes of the femoral growth plate as shown by immunohistochemistry. RT-PCR of in vitro generated mesenchymal stem cells (MSC) and chondrogenically differentiated MSC (cMSC) confirmed the reduced Sfrp1 expression in STR/ort mice. This reduced Sfrp1 expression in MSC correlated with an increased amount of cytoplasmic ß-catenin, a downregulation of the Wnt target gene PPARγ and an upregulation of Runx2 as well as a preferential differentiation of the MSC along the osteoblasts lineage. Given the role of Wnt signalling during chondrogenesis and in maintaining the integrity of the long lived articular chondrocytes, we conclude from our results that the reduced Sfrp1 expression in STR/ort mice not only leads to an increased activation of the Wnt/ß-catenin signalling early in life but also renders the articular cartilage prone to premature ageing and to the development of OA.

The crosstalk between transforming growth factor-ß1 and delta like-1 mediates early chondrogenesis during embryonic endochondral ossification.

Delta like-1 (Dlk1)/preadipocyte factor-1 (Pref-1)/fetal antigen-1 (FA1) is a novel surface marker for embryonic chondroprogenitor cells undergoing lineage progression from proliferation to prehypertrophic stages. However, mechanisms mediating control of its expression during chondrogenesis are not known. Thus, we examined the effect of a number of signaling molecules and their inhibitors on Dlk1 expression during in vitro chondrogenic differentiation in mouse embryonic limb bud mesenchymal micromass cultures and mouse embryonic fibroblast (MEF) pellet cultures. Dlk1/Pref-1 was initially expressed during mesenchymal condensation and chondrocyte proliferation, in parallel with expression of Sox9 and Col2a1, and was downregulated upon the expression of Col10a1 by hypertrophic chondrocytes. Among a number of molecules that affected chondrogenesis, transforming growth factor-ß1 (TGF-ß1)-induced proliferation of chondroprogenitors was associated with decreased Dlk1 expression. This effect was abolished by TGF-ß signaling inhibitor SB431542, suggesting regulation of Dlk1/FA1 by TGF-ß1 signaling in chondrogenesis. TGF-ß1-induced Smad phosphorylation and chondrogenesis were significantly increased in Dlk1(-/-) MEF, while they were blocked in Dlk1 overexpressing MEF, in comparison with wild-type MEF. Furthermore, overexpression of Dlk1 or addition of its secreted form FA1 dramatically inhibited TGF-ß1-induced Smad reporter activity. In conclusion, our data identified Dlk1/FA1 as a downstream target of TGF-ß1 signaling molecule that mediates its function in embryonic chondrogenesis. The crosstalk between TGF-ß1 and Dlk1/FA1 was shown to promote early chondrogenesis during the embryonic endochondral ossification process.

Delta-like 1/fetal antigen-1 (Dlk1/FA1) is a novel regulator of chondrogenic cell differentiation via inhibition of the Akt kinase-dependent pathway.

Delta-like 1 (Dlk1, also known as fetal antigen-1, FA1) is a member of Notch/Delta family that inhibits adipocyte and osteoblast differentiation; however, its role in chondrogenesis is still not clear. Thus, we overexpressed Dlk1/FA1 in mouse embryonic ATDC5 cells and tested its effects on chondrogenic differentiation. Dlk1/FA1 inhibited insulin-induced chondrogenic differentiation as evidenced by reduction of cartilage nodule formation and gene expression of aggrecan, collagen Type II and X. Similar effects were obtained either by using Dlk1/FA1-conditioned medium or by addition of a purified, secreted, form of Dlk1 (FA1) directly to the induction medium. The inhibitory effects of Dlk1/FA1 were dose-dependent and occurred irrespective of the chondrogenic differentiation stage: proliferation, differentiation, maturation, or hypertrophic conversion. Overexpression or addition of the Dlk1/FA1 protein to the medium strongly inhibited the activation of Akt, but not the ERK1/2, or p38 MAPK pathways, and the inhibition of Akt by Dlk1/FA1 was mediated through PI3K activation. Interestingly, inhibition of fibronectin expression by siRNA rescued the Dlk1/FA1-mediated inhibition of Akt, suggesting interaction of Dlk1/FA1 and fibronectin in chondrogenic cells. Our results identify Dlk1/FA1 as a novel regulator of chondrogenesis and suggest Dlk1/FA1 acts as an inhibitor of the PI3K/Akt pathways that leads to its inhibitory effects on chondrogenesis.

Wnt signalling mediates the cross-talk between bone marrow derived pre-adipocytic and pre-osteoblastic cell populations.

The mechanisms underlying the inverse relationship between osteogenic and adipogenic differentiation of bone marrow stromal cells (MSC) are not known in detail. We have previously established two cell lines from mouse bone marrow that are committed to either osteogenic (osteoblasts and chondrocytes) (mMSC(Bone)) or adipogenic (mMSC(Adipo)) lineage. To identify the molecular mechanism determining the lineage commitment, we compared the basal gene expression profile of mMSC(Bone) versus mMSC(Adipo) using Affymetrix GeneChip® MG430A 2.0 Array. Gene annotation analysis based on biological function revealed an over-representation of skeletal development genes in mMSC(Bone) while genes related to lipid metabolism and immune response were highly expressed in mMSC(Adipo). In addition, there was a significant up-regulation of canonical Wnt signalling genes in mMSC(Bone) compared to mMSC(Adipo) (p<0.006). Dual-luciferase assay and expression analysis of genes related to Wnt signalling demonstrated significant activation of Wnt signalling pathway in mMSC(Bone) compared to mMSC(Adipo). Reduced Wnt activity in mMSC(Adipo) was associated with increased expression of the Wnt inhibitor, secreted frizzled-related protein 1 (sFRP-1) at both mRNA and protein levels in mMSC(Adipo). Interestingly, conditioned medium (CM) collected from mMSC(Adipo) (mMSC-CM(Adipo)) inhibited osteoblast differentiation of mMSC, while depletion of sFRP-1 protein from mMSC-CM(Adipo) abolished its inhibitory effect on osteoblast differentiation. Furthermore, treatment of mMSC with recombinant sFRP-1 resulted in a dose-dependent inhibition of osteoblast and stimulation of adipocyte differentiation. In conclusion, cross-talk exists between different populations of MSC in the bone marrow, and Wnt signalling functions as a molecular switch that determines the balance between osteoblastogenesis and adipogenesis.

Isolation and differentiation of chondrocytic cells derived from human embryonic stem cells using dlk1/FA1 as a novel surface marker.

Few surface markers are available to monitor lineage differentiation during chondrogenesis. Recently, delta-like1/fetal antigen1 (dlk1/FA1), a transmembrane protein of the Notch/Delta/Serrata family, was shown to be essential for inducing early chondrogenesis. Thus, we investigated the possible use of dlk1/FA1 as a novel surface marker for chondroprogenitor cells during hESC differentiation. We found that, Dlk1/FA1 is expressed specifically in cells undergoing transition from proliferating to prehypertrophic chondrocytes during endochondral ossification of the mouse limb. In hESC cells, dlk1/FA1 was not expressed by undifferentiated hESC, but expressed during in vitro embryoid bodies (hEBs) formation upon down-regulation of undifferentiated markers e.g. Oct 3/4. Similarly, dlk1/FA1 was expressed in chondrocytic cells during in vivo teratoma formation. Interestingly, treatment of hEBs with Activin B, a member of TGF-ss family, markedly increased Dlk1 expression in association with up-regulation of the mesoderm-specific markers (e.g. FOXF1, KDR and VE-cadherin) and SOX9. dlk1/FA1(+) cells isolated by fluorescence activated cell sorting (FACS) were capable of differentiating into chondrocytic cells when cultured as micromass pellets in a xeno-free system containing TGFbeta1. In conclusion, we identified dlk1/FA1 as a novel marker of chondroprogenitor cells that undergo embryonic lineage progression from proliferation to the prehypertrophic stage. Tracking dlk1/FA1 expression as a mesoderm/chondroprogenitor surface marker provides a novel strategy for designing clinically relevant protocols to direct the differentiation of hESC into chondrocytes.

Impact of stromal cell composition on BMP-induced chondrogenic differentiation of mouse bone marrow derived mesenchymal cells.

Chondrogenic differentiation in mesenchymal stromal cells (MSCs) has been actively studied due to their potential use in mesenchymal tissue repair. Our goal was to develop a simple isolation protocol for adherent mouse MSCs to simultaneously clear off hematopoietic cells and expand to obtain enough starting material for differentiation studies. CD34 and CD45 expressing cells were rapidly removed by inhibiting growth of hematopoietic cells to yield short-term selected (STS) cells. Further passaging enriched more primitive, uniformly Sca-1 expressing, long-term selected (LTS) cells. The efficacy of several BMPs to induce chondrogenesis in pellet culture was compared in STS and LTS cells. In STS cells, chondrogenesis progressed rapidly to terminal differentiation while LTS cells differentiated at a slower rate with no hypertrophy. In LTS cells, rhBMP homodimers -2, -4, -6 and rhBMP2/7 heterodimer were effective enhancers of chondrogenesis over that of rhBMP-5 and -7. In STS cells, rhBMP-2 and rhBMP-7 supported rapid chondrogenesis and terminal differentiation over that of rhBMP-6. These data indicate the impact of stromal cell composition on the chondrogenic differentiation profile, which is an important aspect to be considered when standardizing differentiation assay conditions as well as developing MSC based cartilage repair technologies.

MicroRNAs regulate osteogenesis and chondrogenesis of mouse bone marrow stromal cells.

MicroRNAs (miRNAs) are non-coding RNAs that bind to target mRNA leading to translational arrest or mRNA degradation. To study miRNA-mediated regulation of osteogenesis and chondrogenesis, we compared the expression of 35 miRNAs in osteoblasts and chondroblasts derived from mouse marrow stromal cells (MSCs). Differentiation of MSCs resulted in up- or downregulation of several miRNAs, with miR-199a expression being over 10-fold higher in chondroblasts than in undifferentiated MSCs. In addition, miR-124a was strongly upregulated during chondrogenesis while the expression of miR-96 was substantially suppressed. A systems biological analysis of the potential miRNA target genes and their interaction networks was combined with promoter analysis. These studies link the differentially expressed miRNAs to collagen synthesis and hypoxia, key pathways related to bone and cartilage physiology. The global regulatory networks described here suggest for the first time how miRNAs and transcription factors are capable of fine-tuning the osteogenic and chondrogenic differentiation of mouse MSCs.

Analysis of arthritic lesions in the Del1 mouse: a model for osteoarthritis.

Osteoarthritis (OA) is characterised by progressive erosion of articular cartilage with a number of associated degenerative processes within the joint. Animal models of OA provide the only feasible way to systematically study the development and progression of OA, in order to understand the molecular events, and to develop tools for prevention and therapy of OA. Gene manipulation techniques have provided opportunities to generate transgenic mouse models for OA. In heterozygous Dell mice, incorporation of Col2a1 transgenes with a short deletion mutation results in production of shortened proalpha1 (II) collagen chains and a phenotype resembling human OA. This chapter describes techniques and practical aspects of preparation and processing of skeletal samples for radiological, histological, and molecular biologic analyses that have been used to monitor the development of knee OA in Dell mice. A simple histological grading system to evaluate the progression of OA lesions, and examples of other degenerative alterations in the knee joint structures are presented. Semiquantitative microscopic techniques are described for the analysis of proteoglycan distribution based on safranin O staining of glycosaminoglycans, and for the analysis of collagen matrix based on birefringence of polarized light. Reference is also made to an experimental setup for correlating voluntary running activity of mice with OA score.

Spontaneous development of synovitis and cartilage degeneration in transgenic mice overexpressing cathepsin K.

OBJECTIVE: Several recent studies have demonstrated that cathepsin K, a proteolytic enzyme capable of degrading native fibrillar collagen, is overexpressed in osteoarthritic cartilage and inflamed synovial tissue. However, it is not known whether increased cathepsin K production is a primary or a secondary event in these diseases. The availability of transgenic UTU17 mice, which exhibit constitutive overexpression of the cathepsin K gene, prompted us to study possible arthritic changes in their knee joints. METHODS: Progression of synovitis and articular cartilage degeneration in the knee joints of UTU17 mice and their nontransgenic littermates was monitored by histologic analyses at 7 and 12 months of age. Distribution of cathepsin K in the knee joints was studied by immunohistochemistry. RESULTS: At the age of 7 months, UTU17 mice exhibited clear signs of synovitis, with strong immunostaining for cathepsin K in the synovial lining and the stroma, while control knee joints appeared normal. At 12 months, marked synovial thickening and fibrosis and severe degradation of cartilage and subchondral bone were observed in UTU17 mouse knee joints. In areas of cartilage degeneration, both chondrocytes and cells of hypertrophic synovia were positive for cathepsin K. At 12 months, synovia of control mice revealed only a few isolated cathepsin K-positive cells and mild changes in articular cartilage. CONCLUSION: Our findings demonstrate that overexpression of the cathepsin K gene under its own promoter in transgenic mice makes them susceptible to progressive synovitis, which, upon aging, results in synovial hyperplasia and fibrosis and subsequent destruction of articular cartilage and bone.

The fibril-associated collagen IX provides a novel mechanism for cell adhesion to cartilaginous matrix.

Collagen IX is the prototype fibril-associated collagen with interruptions in triple helix. In human cartilage it covers collagen fibrils, but its putative cellular receptors have been unknown. The reverse transcription-PCR analysis of human fetal tissues suggested that based on their distribution all four collagen receptor integrins, namely alpha1beta1, alpha2beta1, alpha10beta1, and alpha11beta1, are possible receptors for collagen IX. Furthermore primary chondrocytes and chondrosarcoma cells express the four integrins simultaneously. Chondrosarcoma cells, as well as Chinese hamster ovary cells transfected to express alpha1beta1, alpha2beta1, or alpha10beta1 integrin as their only collagen receptor, showed fast attachment and spreading on human recombinant collagen IX indicating that it is an effective cell adhesion protein. To further study the recognition of collagen IX we produced recombinant alphaI domains in Escherichia coli. For each of the four alphaI domains, collagen IX was among the best collagenous ligands, making collagen IX exceptional compared with all other collagen subtypes tested so far. Rotary shadowing electron microscopy images of both alpha1I- and alpha2I-collagen IX complexes unveiled only one binding site located in the COL3 domain close to the kink between it and the COL2 domain. The recognition of collagen IX by alpha2I was considered to represent a novel mechanism for two reasons. First, collagen IX has no GFOGER motif, and the identified binding region lacks any similar sequences. Second, the alpha2I domain mutations D219R and H258V, which both decreased binding to collagen I and GFOGER, had very different effects on its binding to collagen IX. D219R had no effect, and H258V prevented type IX binding. Thus, our results indicate that collagen IX has unique cell adhesion properties when compared with other collagens, and it provides a novel mechanism for cell adhesion to cartilaginous matrix.

Generation and use of transgenic mice as models of osteoarthritis.

The term osteoarthritis (OA) represents a group of diseases characterized by gradual degradation of articular cartilage and a number of associated degenerative processes within the joint. Consequently, no single animal model is likely to fulfil all the criteria of OA. The present chapter discusses the possibilities of using transgenic technologies for modification of the mouse genome to generate animal models of OA. After discussing the different approaches available, we provide an example of the generation of a traditional transgenic mouse strain and describe techniques and practical aspects of genotyping as well as the preparation of skeletal samples for radiological, histological, immunohistological, and molecular biologic analyses for phenotype characterization. We also present a histological grading system to evaluate the progression of OA lesions, with examples of other degenerative alterations in the knee joint structures.

Molecular defect of RAPADILINO syndrome expands the phenotype spectrum of RECQL diseases.

The RECQL4 helicase gene is a member of the RECQL gene family, mutated in some Rothmund-Thomson syndrome (RTS) patients. Other members of this gene family are BLM mutated in Bloom syndrome, WRN mutated in Werner syndrome and RECQL and RECQL5. All polypeptides encoded by RECQL genes share a central region of seven helicase domains. The function of RECQL4 remains unknown, but based on the domain homology it possesses ATP-dependent DNA helicase activity such as BLM and WRN. Rothmund-Thomson, Bloom and Werner syndromes have overlapping clinical features, of which high predisposition to malignancies is the most remarkable feature. Here we report a fourth syndrome resulting in mutations in the RECQL genes. RAPADILINO syndrome is an autosomal recessive disorder characterized by short stature, radial ray defects and other malformations, as well as infantile diarrhoea, but not by a significant cancer risk. Four mutations in the RECQL4 gene were found in the Finnish patients, the most common mutation representing exon 7 in-frame deletion saving the helicase domain and showing dominant effect over other three nonsense mutations. The tissue expression of Recql4 in mouse well agrees with the tissue symptoms of RAPADILINO. The skeletal malformations in RAPADILINO and RTS patients as well as the high osteosarcoma risk in RTS propose a special role for RECQL4 in bone development.

Ultrastructural characterization of developmental and degenerative vitreo-retinal changes in the eyes of transgenic mice with a deletion mutation in type II collagen gene.

PURPOSE: Molecular genetic analyses have clearly associated vitreoretinal degeneration with mutations in the type II collagen gene, but lack of experimental models has prevented systematic analyses of the occurrence of phenotypic changes and of the pathogenetic mechanisms involved. The present study is a detailed morphological and ultrastructural analysis of the vitreoretinal consequences of a small deletion mutation in the type II collagen gene. METHODS: The eyes of Del1 mice carrying six copies of pro alpha1(II) collagen transgene with a small deletion mutation were analyzed during embryonic development, postnatal growth and aging using their nontransgenic littermates as controls. Tissue samples were processed for light and electron microscopy for morphological and ultrastructural analyses. Transcription of pro alpha1(II) collagen gene was localized by in situ hybridization, and type II collagen was detected by immunohistochemistry. RESULTS: In this mouse model most components of the eye are ultrastructurally unaltered. However, the transgenes caused a dose-dependent dominant negative effect seen as a reduced number of type II collagen fibrils in the vitreous. In concert with this, dose-dependent accumulation of amorphous material was observed in the dilated rough endoplasmic reticulum of cells responsible for the production of type II collagen molecules. In mice homozygous for the transgene locus, the vitreoretinal degenerative lesions appeared already during late embryonic development. In mice heterozygous for the locus, such changes were milder and appeared only during postnatal growth and progressed gradually upon aging. CONCLUSIONS: The observed ultrastructural changes suggest that defective structure and function of collagen fibrils in Del1 mice result from a partial block in the post-translational processing and secretion of the mutated procollagen chains, and partly from secretion of mutated procollagen molecules which interfere with normal fibrillogenesis.