TUFT1, a novel candidate gene for metatarsophalangeal osteoarthritis, plays a role in chondrogenesis on a calcium-related pathway

Osteoarthritis (OA) is the most common degenerative joint disorder and genetic factors have been shown to have a significant role in its etiology. The first metatarsophalangeal joint (MTP I) is highly susceptible to development of OA due to repetitive mechanical stress during walking. We used whole exome sequencing to study genetic defect(s) predisposing to familial early-onset bilateral MTP I OA inherited in an autosomal dominant manner. A non-synonymous single nucleotide variant rs41310883 (c.524C>T, p.Thr175Met) in TUFT1 gene was found to co-segregate perfectly with MTP I OA. The role of TUFT1 and the relevance of the identified variant in pathogenesis of MTP I OA were further assessed using functional in vitro analyses. The variant reduced TUFT1 mRNA and tuftelin protein expression in HEK293 cells. ATDC5 cells overexpressing wild type (wt) or mutant TUFT1 were cultured in calcifying conditions and chondrogenic differentiation was found to be inhibited in both cell populations, as indicated by decreased marker gene expression when compared with the empty vector control cells. Also, the formation of cartilage nodules was diminished in both TUFT1 overexpressing ATDC5 cell populations. At the end of the culturing period the calcium content of the extracellular matrix was significantly increased in cells overexpressing mutant TUFT1 compared to cells overexpressing wt TUFT1 and control cells, while the proteoglycan content was reduced. These data imply that overexpression of TUFT1 in ATDC5 inhibits chondrogenic differentiation, and the identified variant may contribute to the pathogenesis of OA by increasing calcification and reducing amount of proteoglycans in the articular cartilage extracellular matrix thus making cartilage susceptible for degeneration and osteophyte formation

Prolyl 4-hydroxylase:genomic cloning of the human and mouse α(II) subunit, tissue distribution of type I and II isoenzymes, and cloning and characterization of a novel prolyl 4-hydroxylase from Caenorhabditis elegans

Abstract The collagens are a family of extracellular matrix proteins with a widespread tissue distribution. Collagen biosynthesis requires the hydroxylation of a number of proline residues by prolyl 4-hydroxylase. This posttranslational modification is essential for the synthesis of all collagens, as 4-hydroxyproline deficient collagens cannot form stable triple helices at body temperature. The genes for the human and mouse prolyl 4-hydroxylase α(II) subunits were cloned and characterized in this study. The human and mouse genes are 34.6 and 30.3 kb in size, respectively, consisting of 16 exons and 15 introns. The intron sizes vary from 48-49 bp to over 8 kb in both genes. The 5' flanking regions contain no TATA box, but there are several motifs that may act as transcription factor binding sites. A novel mutually exclusively spliced exon 12a was identified in both genes. Both variants of the α(II) subunit were found to be expressed in a variety of tissues and both formed a fully active recombinant tetramer with the β subunit when expressed in insect cells. Tissue distribution of the type I and type II prolyl 4-hydroxylase isoenzymes was studied in developing, mature, and malignant cells and tissues by immunofluorescence and Western blotting. The results indicate that the type I isoenzyme is the main form in many cell types. Skeletal myocytes and smooth muscle cells appeared to have the type I isoenzyme as their only prolyl 4-hydroxylase form, whereas the type II isoenzyme was clearly the main form in chondrocytes. A strong signal for the type II enzyme was detected in cultured umbilical and capillary endothelial cells, whereas the type I isoenzyme could not be detected in these cells by immunostaining or Western blotting. Similar studies on primary chondro- and osteosarcomas and benign bone tumours indicated that the type I isoenzyme is the predominant form in both types of bone sarcoma, whereas the type II isoenzyme was more abundantly expressed in benign tumours. In chondrosarcomas, the type II isoenzyme was expressed in the nonmalignant chondrocytes, whereas their malignant counterparts switched their expression pattern to that of the type I isoenzyme. Two isoforms of the catalytic prolyl 4-hydroxylase α subunit, PHY-1 and PHY-2, have previously been characterized from Caenorhabditis elegans. This study reports the cloning and characterization of a third C. elegans α subunit isoform, PHY-3, which is much shorter than the previously characterized vertebrate and C. elegans α subunits. Nematodes homozygous for a phy-3 deletion were phenotypically wild type and fertile, but the 4-hydroxyproline content of their early embryos was reduced by about 90%. The expression of PHY-3 was found to be restricted to spermatheca of late larvae and adult nematode, indicating that PHY-3 is likely to be involved in the synthesis of collagens of the early embryo egg shells

Histological findings of patients with adnexal torsion who underwent surgical treatment:short reminder

Abstract Background: Ovarian torsion is a rare emergency condition in women. Early diagnosis is necessary to preserve fertility. Case: Our study evaluated 40 patients, who underwent laparoscopic surgery. The aims of this retrospective study were to emphasize the importance of early diagnosis in ovarian torsion, evaluate the process of patient treatment, and investigate the number of patients treated by minimally invasive surgery. In this article, we present the outcomes from the patient data. Results: Thirty-two percent (13/40) of patients were first evaluated by the surgeon to investigate right-sided lower abdominal pain. These patients were first misdiagnosed with appendicitis or urinary tract stones. Among these patients, necrotic ovary tissue was more common, most likely due to a longer delay seeking medical attention. A total of 77% (31/40) of patients underwent laparoscopic surgery on the same date that they were admitted to the hospital. No severe complications occurred in this group of patients. All histological findings were benign. In 52% (21/40) of patients, the adnexa was removed, whereas in 37% (15/40) of patients the torsed adnexa was treated by detorsion. A total of 27% (11/40) of patients had no diagnosis before undergoing surgery. Conclusions: Rapid and accurate diagnosis is essential to preserve ovarian function

The serum levels of circulating matrix metalloproteinase MMP-9, MMP-2/TIMP-2 complex and TIMP-1 do not change significantly during normal pregnancy:a pilot study

Abstract Objective: Matrix metalloproteinases (MMPs) are important regulators of vascular and uterine remodeling. They exhibit proteolytic activity implicating the efficiency of trophoblast invasion to the uterine wall involving marked hemodynamic and uterine changes. In this pilot study sera of 13 women with normal pregnancy was analyzed to evaluate the usage of MMPs as diagnostic tool. The concentrations of circulating MMP-9, MMP-2/TIMP-2 complex and TIMP-1 in different time points during normal pregnancy has not been studied. The serum levels of MMP-9, TIMP-1, TIMP-2 and MMP-2/TIMP-2 complex were determined by enzyme-linked immunosorbent assay (ELISA). Using the same method, we have shown that serum MMPs are elevated in spontaneous early pregnancy failure as compared to normal pregnancy. Results: The serum levels of MMP-9 and TIMP-1 were stable throughout pregnancy. The level of MMP-2/TIMP-2 complex was slightly increased after week 15 without statistical significance. For our best knowledge, this is a first study of the serum levels of MMP-9, MMP-2/TIMP-2 and TIMP-1 on different time points during normal pregnancy. Further measurements with the correlation to the outcome of the pregnancy are needed

Circulating matrix metalloproteinase MMP-9 and MMP-2/TIMP-2 complex are associated with spontaneous early pregnancy failure

Abstract Background Trophoblast cell (CTB) invasion into the maternal endometrium plays a crucial role during human embryo implantation and placentation. This invasion is facilitated by the activity of matrix metalloproteinases, which are regulated by tissue inhibitors of MMPs (TIMPs). Methods This study compares the serum levels of MMP-9, MMP-2/TIMP-2 complex, TIMP-1 and TIMP-2 in 129 patients with ongoing pregnancy (n = 40) or spontaneous early pregnancy failure (n = 89). Results MMP-9 was markedly (p  Conclusions Human placentation is mediated by fetal trophoblastic cells that invade the maternal uterine endometrium. Trophoblast invasion requires a precisely regulated secretion of specific proteolytic enzymes able to degrade the endometrial basement membrane and extracellular matrix. The elevated levels of MMP-9 and MMP-2/TIMP-2 complex may play a role in spontaneous termination of pregnancy.</p

TUFT1, a novel candidate gene for metatarsophalangeal osteoarthritis, plays a role in chondrogenesis on a calcium-related pathway

Abstract Osteoarthritis (OA) is the most common degenerative joint disorder and genetic factors have been shown to have a significant role in its etiology. The first metatarsophalangeal joint (MTP I) is highly susceptible to development of OA due to repetitive mechanical stress during walking. We used whole exome sequencing to study genetic defect(s) predisposing to familial early-onset bilateral MTP I OA inherited in an autosomal dominant manner. A nonsynonymous single nucleotide variant rs41310883 (c.524C&gt;T, p.Thr175Met) in TUFT1 gene was found to co-segregate perfectly with MTP I OA. The role of TUFT1 and the relevance of the identified variant in pathogenesis of MTP I OA were further assessed using functional in vitro analyses. The variant reduced TUFT1 mRNA and tuftelin protein expression in HEK293 cells. ATDC5 cells overexpressing wild type (wt) or mutant TUFT1 were cultured in calcifying conditions and chondrogenic differentiation was found to be inhibited in both cell populations, as indicated by decreased marker gene expression when compared with the empty vector control cells. Also, the formation of cartilage nodules was diminished in both TUFT1 overexpressing ATDC5 cell populations. At the end of the culturing period the calcium content of the extracellular matrix was significantly increased in cells overexpressing mutant TUFT1 compared to cells overexpressing wt TUFT1 and control cells, while the proteoglycan content was reduced. These data imply that overexpression of TUFT1 in ATDC5 inhibits chondrogenic differentiation, and the identified variant may contribute to the pathogenesis of OA by increasing calcification and reducing amount of proteoglycans in the articular cartilage extracellular matrix thus making cartilage susceptible for degeneration and osteophyte formation