tenascin-X Research Articles

Tenascin-X in amniotic fluid and reproductive tissues of pregnancies complicated by infection and preterm prelabor rupture of membranes†.

Preterm prelabor rupture of membranes (PPROM), which can precede or follow intra-amniotic infection/inflammation (IAI), is a poorly understood pregnancy complication. Tenascin-X (TNX) is a connective tissue extracellular matrix protein that regulates fibrillogenesis of collagens I, III, and V. Our goal was to investigate the presence and level of soluble TNX (sTNX) in amniotic fluid (AF) and TNX expression in reproductive tissues of pregnancies complicated by PPROM and IAI. We prospectively recruited 334 women pregnant with singletons who had a clinically indicated amniocentesis for genetic karyotyping, lung maturity testing, or rule-out IAI in the presence or absence of PPROM. We quantified TNX expression in fetal membranes, myometrium, cervix, and placenta using immunological methods and qRT-PCR. In pregnancies with normal outcomes, AF sTNX levels were GA-regulated with lower levels toward term. IAI significantly upregulated AF sTNX levels independent of membrane status. AF sTNX levels inversely correlated with fetal membranes tenascin XB (TNXB) mRNA level, which was significantly downregulated by IAI. Western blotting identified characteristic ∼75 and ∼140 kDa sTNX forms in both AF and fetal membranes. Fetal membranes, placenta, and cervix constitutively express TNX with the highest abundance in the amnion. Amnion TNX richness is significantly lost in the setting of IAI. Our results suggest that fetal membranes may be a source of AF sTNX whereby protein and mRNA expression seem to be significantly impacted by inflammation independent of fetal membrane status. A more thorough understanding of TNX changes may be valuable for understanding spontaneous PPROM and to potentially develop therapeutic targets.

The extracellular matrix glycoprotein tenascin-X regulates peripheral sensory and motor neurones.

Key points Tenascin‐X (TNX) is an extracellular matrix glycoprotein with anti‐adhesive properties in skin and joints. Here we report the novel finding that TNX is expressed in human and mouse gut tissue where it is exclusive to specific subpopulations of neurones.Our studies with TNX‐deficient mice show impaired defecation and neural control of distal colonic motility that can be rescued with a 5‐HT4 receptor agonist. However, colonic secretion is unchanged. They are also susceptible to internal rectal intussusception. Colonic afferent sensitivity is increased in TNX‐deficient mice. Correspondingly, there is increased density of and sensitivity of putative nociceptive fibres in TNX‐deficient mucosa.A group of TNX‐deficient patients report symptoms highly consistent with those in the mouse model.These findings suggest TNX plays entirely different roles in gut to non‐visceral tissues – firstly a role in enteric motor neurones and secondly a role influencing nociceptive sensory neuronesStudying further the mechanisms by which TNX influences neuronal function will lead to new targets for future treatment. The extracellular matrix (ECM) is not only an integral structural molecule, but is also critical for a wide range of cellular functions. The glycoprotein tenascin‐X (TNX) predominates in the ECM of tissues like skin and regulates tissue structure through anti‐adhesive interactions with collagen. Monogenic TNX deficiency causes painful joint hypermobility and skin hyperelasticity, symptoms characteristic of hypermobility Ehlers Danlos syndrome (hEDS). hEDS patients also report consistently increased visceral pain and gastrointestinal (GI) dysfunction. We investigated whether there is a direct link between TNX deficiency and GI pain or motor dysfunction. We set out first to learn where TNX is expressed in human and mouse, then determine how GI function, specifically in the colon, is disordered in TNX‐deficient mice and humans of either sex. In human and mouse tissue, TNX was predominantly associated with cholinergic colonic enteric neurones, which are involved in motor control. TNX was absent from extrinsic nociceptive peptidergic neurones. TNX‐deficient mice had internal rectal prolapse and a loss of distal colonic contractility which could be rescued by prokinetic drug treatment. TNX‐deficient patients reported increased sensory and motor GI symptoms including abdominal pain and constipation compared to controls. Despite absence of TNX from nociceptive colonic neurones, neuronal sprouting and hyper‐responsiveness to colonic distension was observed in the TNX‐deficient mice. We conclude that ECM molecules are not merely support structures but an integral part of the microenvironment particularly for specific populations of colonic motor neurones where TNX exerts functional influences.

Multiple Roles of Tenascins in Homeostasis and Pathophysiology of Aorta.

Tenascins are a family of large extracellular matrix (ECM) glycoproteins. Four family members (tenascin-C, -R, -X, and -W) have been identified to date. Each member consists of the same types of structural domains and exhibits time- and tissue-specific expression patterns, suggesting their specific roles in embryonic development and tissue remodeling. Among them, the significant involvement of tenascin-C (TNC) and tenascin-X (TNX) in the progression of vascular diseases has been examined in detail. TNC is strongly up-regulated under pathological conditions, induced by a number of inflammatory mediators and mechanical stress. TNC has diverse functions, particularly in the regulation of inflammatory responses. Recent studies suggest that TNC is involved in the pathophysiology of aneurysmal and dissecting lesions, in part by protecting the vascular wall from destructive mechanical stress. TNX is strongly expressed in vascular walls, and its distribution is often reciprocal to that of TNC. TNX is involved in the stability and maintenance of the collagen network and elastin fibers. A deficiency in TNX results in a form of Ehlers–Danlos syndrome (EDS). Although their exact roles in vascular diseases have not yet been elucidated, TNC and TNX are now being recognized as promising biomarkers for diagnosis and risk stratification of vascular diseases.

Selective Mineralocorticoid Receptor Cofactor Modulation as Molecular Basis for Finerenone's Antifibrotic Activity.

Mineralocorticoid receptor antagonists (MRAs) reduce morbidity and mortality in chronic heart failure. Novel nonsteroidal MRAs are currently developed and need to be pharmacologically characterized in comparison to classical steroidal MRAs. A mouse model of cardiac fibrosis induced by short-term isoproterenol injection was used to compare the nonsteroidal MRA finerenone and the steroidal MRA eplerenone in equi-efficient systemic MR blocking dosages. Molecular mechanisms were studied in MR-expressing H9C2/MR+ cardiomyocytes and in MR transcriptional cofactor binding assays. Both MRAs significantly inhibited an isoproterenol-mediated increase of left ventricular mass. Isoproterenol-induced cardiac fibrosis and macrophage invasion were potently blocked by finerenone, whereas eplerenone had no significant effect. Speckle tracking echocardiography revealed a significant improvement of global longitudinal peak strain by finerenone, an effect less prominent with eplerenone. Antifibrotic actions of finerenone were accompanied by a significant inhibition of profibrotic cardiac TNX (tenascin-X) expression, a regulation absent with eplerenone. Finally, we show a higher potency/efficacy and inverse agonism of finerenone versus eplerenone in MR transcriptional cofactor binding assays indicating differential MR cofactor modulation by steroidal and nonsteroidal MRAs. This study demonstrates that the nonsteroidal MRA finerenone potently prevents cardiac fibrosis and improves strain parameters in mice. Cardiac antifibrotic actions of finerenone may result from the inhibition of profibrotic TNX gene expression mediated by differential MR cofactor binding. Selective MR cofactor modulation provides a molecular basis for distinct (pre)-clinical actions of nonsteroidal and steroidal MRAs.

Wound healing-related properties detected in an experimental model with a collagen gel contraction assay are affected in the absence of tenascin-X

Patients with tenascin-X (TNX)-deficient type Ehlers-Danlos syndrome (EDS) do not exhibit delayed wound healing, unlike classic type EDS patients, who exhibit mutations in collagen genes. Similarly, in TNX-knockout (KO) mice, wound closure of the skin is normal even though these mice exhibit a reduced breaking strength. Therefore, we speculated that the wound healing process may be affected in the absence of TNX. In this study, to investigate the effects of TNX absence on wound healing-related properties, we performed collagen gel contraction assays with wild-type (WT) and TNX-KO mouse embryonic fibroblasts (MEFs). Collagen gels with embedded TNX-KO MEFs showed significantly greater contraction than those containing WT MEFs. Subsequently, we assessed collagen gel contraction-related properties, such as the activities of matrix metalloproteinase (MMP)-2 and MMP-9 and the protein and mRNA expression levels of transforming growth factor β1 (TGF-β1) in the collagen gels. The activities of MMP-2 and MMP-9 and the expression level of TGF-β1 were elevated in the absence of TNX. Furthermore, filopodia-like protrusion formation, cell proliferation, migration, and collagen expression in MEFs were promoted in the absence of TNX. These results indicate that these wound healing-related properties are affected in a TNX-deficient extracellular environment.

Smoking is associated with hypermethylation of the APC 1A promoter in colorectal cancer: the ColoCare Study.

Smoking tobacco is a known risk factor for the development of colorectal cancer and for mortality associated with the disease. Smoking has been reported to be associated with changes in DNA methylation in blood and in lung tumour tissues, although there has been scant investigation of how epigenetic factors may be implicated in the increased risk of developing colorectal cancer. To identify epigenetic changes associated with smoking behaviours, we performed epigenome-wide analysis of DNA methylation in colorectal tumours from 36 never-smokers, 47 former smokers, and 13 active smokers, and in adjacent mucosa from 49 never-smokers, 64 former smokers, and 18 active smokers. Our analyses identified 15 CpG sites within the APC 1A promoter that were significantly hypermethylated and 14 CpG loci within the NFATC1 gene body that were significantly hypomethylated (pLIS < 1 × 10-5 ) in the tumours of active smokers. The APC 1A promoter was hypermethylated in 7 of 36 tumours from never-smokers (19%), 12 of 47 tumours from former smokers (26%), and 8 of 13 tumours from active smokers (62%). Promoter hypermethylation was positively associated with duration of smoking (Spearman rank correlation, ρ = 0.26, p = 0.03) and was confined to tumours, with hypermethylation never being observed in adjacent mucosa. Further analysis of adjacent mucosa revealed significant hypomethylation of four loci associated with the TNXB gene in tissue from active smokers. Our findings provide exploratory evidence for hypermethylation of the key tumour suppressor gene APC being implicated in smoking-associated colorectal carcinogenesis. Further work is required to establish the validity of our observations in independent cohorts. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Suppression of hepatic dysfunction in tenascin‑X‑deficient mice fed a high‑fat diet.

Extracellular matrix glycoprotein tenascin-X (TNX) is the largest member of the tenascin family. In the present study, the contribution of TNX to liver dysfunction was investigated by administration of high-fat and high-cholesterol diet with high levels of phosphorus and calcium (HFCD) to wild-type (WT) and TNX-knockout (KO) mice. After 16 weeks of HFCD administration, the ratio of liver weight to body weight was approximately 22% higher in the HFCD-fed WT mice compared with the HFCD-fed TNX-KO mice, indicating hepatomegaly in HFCD-fed WT mice. Histological analyses with hematoxylin and eosin staining at 21 weeks revealed that hepatocyte hypertrophy in HFCD-fed TNX-KO mice was suppressed to 85% of that in HFCD-fed WT mice. By contrast, there was a 1.2-fold increase in lipid deposition in hepatocytes from HFCD-fed TNX-KO mice compared with HFCD-fed WT mice at 18 weeks, as demonstrated by Oil Red O staining. In addition, TNX-KO mice at 21 weeks and 27 weeks post-HFCD administration exhibited significant suppression of inflammatory cell infiltrate to 51 and 24% of that in WT mice, respectively. Immunofluorescence analysis for type I collagen and Elastica van Gieson staining demonstrated a clear hepatic fibrosis progression in HFCD-fed WT mice at 27 weeks, whereas hepatic fibrosis was undetected in HFCD-fed TNX-KO mice. The present findings indicated that TNX deficiency suppressed hepatic dysfunction induced by HFCD administration.

A potential contribution of tenascin-X to blood vessel formation in peripheral nerves

Tenascin-X (TNX), an extracellular matrix protein, is abundantly expressed in peripheral nerves. However, the physiological role of TNX in peripheral nerves remains unknown. In this study, we found that actin levels in sciatic nerves of TNX-deficient mice were markedly decreased. Since actin was highly expressed in endothelial cells in wild-type sciatic nerves, we assessed morphological alterations of blood vessels in TNX-null sciatic nerves. The density of blood vessels was significantly decreased and the size of blood vessels was larger than those in wild-type sciatic nerves. Immunofluorescence showed that TNX was expressed by Schwann cells and fibroblasts in sciatic nerves. The results suggest that TNX secreted from Schwann cells and/or fibroblasts is involved in blood vessel formation in peripheral nerves.

Genetic similarities and differences between discoid and systemic lupus erythematosus patients within the Polish population.

IntroductionMany studies have shown that some SNPs might be a risk factor for systemic lupus erythematosus (SLE), but little is known about potential susceptibility loci of the skin types of the disease. Discoid lupus erythematosus (DLE) is the most common form of the cutaneous lupus erythematosus. Nevertheless, a genetic contribution to DLE is not fully recognized.AimWe aimed to analyze three SNPs located in the STAT4 (rs7574865), ITGAM (rs1143679) and TNXB (rs1150754) genes in both DLE and SLE patients from Poland.Material and methodsSNPs were genotyped using real-time polymerase chain reaction (PCR). Statistical significance of the differences between patient and control groups in both allele and genotype frequencies were calculated using two tailed Fisher’s exact test. The correction for multiple testing by the Bonferroni adjustment and odds ratio were also calculated.ResultsFor the first time, we have shown that the polymorphisms located in the STAT4 (rs7574865), but not in the ITGAM (rs1143679) nor the TNXB (rs1150754) genes, might be associated with the development of DLE within the Polish population. The variation of the three investigated SNPs was found to be associated with SLE in our dataset.ConclusionsThe results of our study suggest differences in the molecular background between DLE and SLE within the Polish population.

Recognizing the tenascin-X deficient type of Ehlers-Danlos syndrome: a cross-sectional study in 17 patients.

The tenascin-X (TNX) deficient type Ehlers-Danlos syndrome (EDS) is similar to the classical type of EDS. Because of the limited awareness among geneticists and the challenge of the molecular analysis of the TNXB gene, the TNX-deficient type EDS is probably to be under diagnosed. We therefore performed an observational, cross-sectional study. History and physical examination were performed. Results of serum TNX measurements were collected and mutation analysis was performed by a combination of next-generation sequencing (NGS), Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA). Included were 17 patients of 11 families with autosomal recessive inheritance and childhood onset. All patients had hyperextensible skin without atrophic scarring. Hypermobility of the joints was observed in 16 of 17 patients. Deformities of the hands and feet were observed frequently. TNX serum level was tested and absent in 11 patients (seven families). Genetic testing was performed in all families; 12 different mutations were detected, most of which are suspected to lead to non-sense mRNA mediated decay. In short, patients with the TNX-deficient type EDS typically have generalized joint hypermobility, skin hyperextensibility and easy bruising. In contrast to the classical type, the inheritance pattern is autosomal recessive and atrophic scarring is absent. Molecular analysis of TNXB in a diagnostic setting is challenging.

A method for quantification of serum tenascin-X by nano-LC/MS/MS

BackgroundComplete deficiency of an extracellular matrix tenascin-X (TNX) leads to a classical type of Ehlers-Danlos syndrome (EDS). TNX haploinsufficiency is a cause of hypermobility type of EDS. Human TNX is also present in a serum form (sTNX) with a molecular size of 140kDa. In this study, we established a method for quantification of sTNX using nano-liquid chromatography tandem mass spectrometry (LC/MS/MS) with selected/multiple reaction monitoring. MethodsTwelve abundant protein-depleted sera were reduced, alkylated, and digested with Lys-C and trypsin. Subsequently, the digests were fractionated by strong cation exchange chromatography. Optimal and validated transitions of precursor and product ions of the peptides from sTNX were developed on a triple quadrupole mass spectrometer. ResultsSerum concentrations of sTNX of healthy individuals were quantified as an average of 144ng/ml. However, sTNX was not detected by this method in serum from a patient with a classical type of EDS in whom sTNX was not found by Western blot analysis. The limit of quantification (LOQ) of sTNX by nano-LC/MS/MS method was 2.8pg whereas the detection sensitivity of sTNX by Western blot analysis was 19pg. The nano-LC/MS/MS method is more sensitive than Western blot analysis. ConclusionsThe quantification method will be useful for diagnosis and risk stratification of EDS caused by TNX deficiency and haploinsufficiency.

Global proteome profiling of dental cementum under experimentally-induced apposition

Dental cementum (DC) covers the tooth root and has important functions in tooth attachment and position. DC can be lost to disease, and regeneration is currently unpredictable due to limited understanding of DC formation. This study used a model of experimentally-induced apposition (EIA) in mice to identify proteins associated with new DC formation. Mandibular first molars were induced to super-erupt for 6 and 21days after extracting opposing maxillary molars. Decalcified and formalin-fixed paraffin-embedded mandible sections were prepared for laser capture microdissection. Microdissected protein extracts were analyzed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), and the data submitted to repeated measure ANOVA test (RM-ANOVA, alpha=5%). A total of 519 proteins were identified, with 97 (18.6%) proteins found exclusively in EIA sites and 50 (9.6%) proteins exclusively expressed in control sites. Fifty six (10.7%) proteins were differentially regulated by RM-ANOVA (p<0.05), with 24 regulated by the exclusive effect of EIA (12 proteins) or the interaction between EIA and time (12 proteins), including serpin 1a, procollagen C-endopeptidase enhancer, tenascin X (TNX), and asporin (ASPN). In conclusion, proteomic analysis demonstrated significantly altered protein profile in DC under EIA, providing new insights on DC biology and potential candidates for tissue engineering applications. SignificanceDental cementum (DC) is a mineralized tissue that covers the tooth root surface and has important functions in tooth attachment and position. DC and other periodontal tissues can be lost to disease, and regeneration is currently unpredictable due to lack of understanding of DC formation. This study used a model of experimentally-induced apposition (EIA) in mice to promote new cementum formation, followed by laser capture microdissection (LCM) and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) proteomic analysis. This approach identified proteins associated with new cementum formation that may be targets for promoting cementum regeneration.

Identification of Biomarkers for Footpad Dermatitis Development and Wound Healing.

Footpad dermatitis (FPD) is a type of skin inflammation that causes necrotic lesions on the plantar surface of the footpads in commercial poultry, with significant animal welfare, and economic implications. To identify biomarkers for FPD development and wound healing, a battery cage trial was conducted in which a paper sheet was put on the bottom of cages to hold feces to induce FPD of broilers. Day-of-hatch Ross 308 male broiler chicks were fed a corn–soybean meal diet and assigned to 3 treatments with 8 cages per treatment and 11 birds per cage. Cages without paper sheets were used as a negative control (NEG). Cages with paper sheets during the entire growth period (d 0–30) were used as a positive control (POS) to continually induce FPD. Cages with paper sheets during d 0–13 and without paper sheets during d 14–30 were used to examine the dynamic of FPD development and lesion wound healing (LWH). Footpad lesions were scored to grade (G) 1–5 with no lesion in G1 and most severe lesion in G5. Covering with paper sheets in POS and LWH induced 99% incidence of G3 footpads on d 13. Removing paper sheets from LWH healed footpad lesions by d 30. One representative bird, with lesions most close to pen average lesion score, was chosen to collect footpad skin samples for biomarker analysis. Total collagen protein and mRNA levels of tenascin X (TNX), type I α1 collagen (COL1A1), type III α1 collagen (COL3A1), tissue inhibitor of metalloproteinase 3 (TIMP3), and integrin α1 (ITGA1) mRNA levels were decreased (P < 0.05), while mRNA levels of tenascin C (TNC), tumor necrosis factor (TNF) α, Toll-like receptor (TLR) 4 and vascular endothelial growth factor (VEGF), IL-1β, and the ratio of MMP2 to all TIMP were increased (P < 0.03) in G3 footpads in POS and LWH compared to G1 footpads in NEG on d 14. These parameters continued to worsen with development of more severe lesions in POS. After paper sheets were removed (i.e., LWH), levels of these parameters gradually or rapidly returned to levels measured in NEG. Regression analysis indicated significant quadratic changes of these parameters to footpad lesion scores. In summary, these biomarkers were interrelated with dynamic changes of footpad lesion scores, suggesting they may be used as potential biomarkers for footpad lesion development and wound healing process.

Mutation in TNXB gene causes moderate to severe Ehlers-Danlos syndrome.

We report a 28-year-old female who presented with severe joint pain, chronic muscle weakness, Raynaud’s phenomenon, and hypermobility. She was found to have a 6074A > T nucleotide transition in the TNXB gene causing an amino acid protein change at Asp2025Val classified as likely pathogenic. We add this clinical report to the literature and classical human disease gene catalogs to identify this specific mutation as disease-causing. This gene variant was reported previously in a different 36-year-old patient who shared our patient’s symptoms of joint hypermobility, skeletal and joint pain, skin elasticity and musculoskeletal problems, thereby causing a more severe presentation than seen in the hypermobility type of Ehlers-Danlos syndrome (EDS). At the time of writing, a few mutations in the TNXB gene have been recognized as pathogenic causing EDS due to tenascin-X deficiency, but the variant identified in our patient has not been recognized as pathogenic in online genetic databases. Our case study in combination with peer-reviewed literature suggests that the 6074A > T nucleotide transition in the TNXB gene may be classified as disease-causing for EDS due to tenascin-X deficiency.

Broadening the Spectrum of Ehlers Danlos Syndrome in Patients With Congenital Adrenal Hyperplasia.

The contiguous gene deletion syndrome (CAH-X) was described in a subset (7%) of congenital adrenal hyperplasia (CAH) patients with a TNXA/TNXB chimera, resulting in deletions of CYP21A2, encoding 21-hydroxylase necessary for cortisol biosynthesis, and TNXB, encoding the extracellular matrix glycoprotein tenascin-X (TNX). This TNXA/TNXB chimera is characterized by a 120-bp deletion in exon 35 and results in TNXB haploinsufficiency, disrupted TGF-β signaling, and an Ehlers Danlos syndrome phenotype. The objective of the study was to determine the genetic status of TNXB and resulting protein defects in CAH patients with a CAH-X phenotype but not the previously described TNXA/TNXB chimera. Design, Settings, Participants, and Intervention: A total of 246 unrelated CAH patients were screened for TNXB defects. Genetic defects were investigated by Southern blotting, multiplex ligation-dependent probe amplification, Sanger, and next-generation sequencing. Dermal fibroblasts and tissue were used for immunoblotting, immunohistochemical, and coimmunoprecipitation experiments. The genetic and protein status of tenascin-X in phenotypic CAH-X patients was measured. Seven families harbor a novel TNXB missense variant c.12174C>G (p.C4058W) and a clinical phenotype consistent with hypermobility-type Ehlers Danlos syndrome. Fourteen CAH probands carry previously described TNXA/TNXB chimeras, and seven unrelated patients carry the novel TNXB variant, resulting in a CAH-X prevalence of 8.5%. This highly conserved pseudogene-derived variant in the TNX fibrinogen-like domain is predicted to be deleterious and disulfide bonded, results in reduced dermal elastin and fibrillin-1 staining and altered TGF-β1 binding, and represents a novel TNXA/TNXB chimera. Tenascin-X protein expression was normal in dermal fibroblasts, suggesting a dominant-negative effect. CAH-X syndrome is commonly found in CAH due to 21-hydroxylase deficiency and may result from various etiological mechanisms.

Modifying Other’s Originality without Quote is an Act of Piracy

A defective CYP21A2 gene downstream of the TNXB gene in congenital adrenal hyperplasia (CAH) falls into three categories: (a) small-scale conversions of CYP21A1P, (b) spontaneous mutations, and (c) chimeric RCCX modules that include the chimeric CYP21A1P/ CYP21A2 and TNXA/TNXB genes [1]. Most of the CYP21A2 mutations identified so far were a result of small-scale conversions of the CYP21A1P (up to 11 for CYP21A1P) during both meiosis and mitosis [2], which account for about 70%-80% of all CAH cases. The Chimeric CYP21A1P/CYP21A2 and TNXA/TNXB genes, which result from unequal cross-over (or deletions) during meiosis [2] and occur in ~20% of CAH alleles in most populations [1,3] respectively reflect the deletion of the 1/XCYP21A1P - XA - RP2 - C4B - 1/XCYP21A2 gene array (1/X indicates an uncertain fraction of the gene sequence) [1] and a deletion of the RP2 - C4B - CYP21A2 - 1/XTXNB gene array [1]. Their deletion range is about a 26- or 32-kb gene sequence which depends on whether C4B is the long or short gene (more commonly shown in the literature as being 30 kb). In fact, these different types of large-gene deletions in the RCCX region are generally considered to represent one event in many studies.

Lack of genotypephenotype correlation in congenital adrenal hyperplasia due to a CYP21A2-like gene

ContextCongenital Adrenal Hyperplasia (CAH) due to 21-hydroxylase deficiency, encoded by CYP21A2 gene, is an autosomal recessive disorder. The CYP21A2 gene, localized in a genetic unit defined RCCX module, is considered one of the most polymorphic of human genes. ObjectivesWe considered new evidences about the presence of a RCCX trimodular haplotype with a CYP21A2-like gene to explain the lack of a genotype–phenotype correlation in individuals of two different families. Design and methodsTo verify gene duplication we used Multiplex Ligation Probe-Dependent Amplifications (MLPA) and to confirm the presence of a CYP21A2-like gene downstream TNXA gene we used previously described amplification and restriction strategy followed by the sequencing of the CYP21A2 gene downstream TNXB gene. ResultsThe amplification strategy and restriction analysis of CYP21A1P/CYP21A2-TNXA PCR product in association with MLPA assay and sequencing of CYP21A2 gene downstream TNXB were able to identify the presence of the CYP21A2-like gene in healthy subjects of the two families, wherein the direct sequencing of CYP21A2 gene showed genotypes correlated to pathological phenotypes. ConclusionsThe strategy suggested is useful to facilitate molecular testing in CAH patients, considering the new evidence about possible different haplotypes.

Tenascin-X promotes epithelial-to-mesenchymal transition by activating latent TGF-β.

Transforming growth factor β (TGF-β) isoforms are secreted as inactive complexes formed through noncovalent interactions between the bioactive TGF-β entity and its N-terminal latency-associated peptide prodomain. Extracellular activation of the latent TGF-β complex is a crucial step in the regulation of TGF-β function for tissue homeostasis. We show that the fibrinogen-like (FBG) domain of the matrix glycoprotein tenascin-X (TNX) interacts physically with the small latent TGF-β complex in vitro and in vivo, thus regulating the bioavailability of mature TGF-β to cells by activating the latent cytokine into an active molecule. Activation by the FBG domain most likely occurs through a conformational change in the latent complex and involves a novel cell adhesion-dependent mechanism. We identify α11β1 integrin as a cell surface receptor for TNX and show that this integrin is crucial to elicit FBG-mediated activation of latent TGF-β and subsequent epithelial-to-mesenchymal transition in mammary epithelial cells.

Molecular analysis of the CYP21A2 gene in Chinese patients with steroid 21-hydroxylase deficiency

Objective21-Hydroxylase deficiency (21-OHD) is the most common cause of congenital adrenal hyperplasia (CAH), a family of autosomal recessive disorders involving impaired cortisol synthesis. This study aimed to design a reliable and rational approach for identifying mutations in the CYP21A2 gene and to characterize the molecular basis of 21-OHD in 30 Chinese patients. Design and methodsCopy number variations were investigated by multiplex ligation-dependent probe amplification (MLPA). Locus-specific polymerase chain reaction (PCR)/restriction endonuclease analysis was then used to verify CYP21A2 rearrangement products and prevent allele dropout. Direct sequencing of rearrangement products was performed to further refine recombination breakpoint locations. Direct sequencing of the entire CYP21A2 gene was used to detect microconversions. ResultsWe successfully characterized 60 CYP21A2 alleles from 30 patients with genetic defects. The most common one was intron 2 splice mutation (38.3%). Eighteen alleles with large gene deletions/conversions were identified, which accounted for nearly one-third (30.0%) of the genetic defects. Among these, three types of CYP21A1P/CYP21A2 chimeric genes (CH-1, CH-2, and CH-4) were characterized. Two novel CYP21A2 rearrangement genes were revealed and further demonstrated to be located downstream of the TNXB gene. ConclusionsOur results indicate that the stepwise diagnostic procedure involving MLPA analysis, locus-specific PCR/restriction endonuclease analysis, and direct DNA sequencing can provide detailed genetic information about Chinese 21-OHD patients, which is helpful for characterizing structural rearrangements of CYP21A2.

O.2 Collagen type XII: A new congenital matrix and muscle disease

In a small proportion of patients with an Ullrich-like clinical phenotype no mutations in the collagen VI genes are detected. Here we report on a novel muscle and tendon collagenopathy caused by mutations in COL12A1. In a consanguineous family with hypotonia, weakness and significant joint hyperlaxity we identified a homozygous frame shifting mutation in COL12A1 in the two affected children, causing complete absence of collagen XII in muscle and fibroblasts. Patients were unable to walk and developed scoliosis and respiratory insufficiency. In the second family the proband presented with milder hypotonia, motor delay, hyperlaxity and incomplete deficiency of collagen XII. We identified a de novo, presumably dominantly acting missense mutation. COL12A1 encodes collagen XII. Collagen XII is a homotrimer found in association with collagen I and acts as a cross-bridge between collagen fibrils while also interacting with tenascin X (TNX), heparin and decorin. We next investigated the muscle phenotype in a Col12a1 KO mouse, which showed age dependent weakness. At 9 months of age there was no significant difference in muscle fiber size, but the overall weight of the muscles was less with an altered slow-to-fast fiber type transition in both soleus and tibilias anterior muscles. Physiological measurements on isolated EDL showed protection from eccentric force drop and a decrease in passive force, suggesting an increased compliance of the matrix as well as decreased lateral and longitudinal force transmission to the matrix and tendon. We hypothesize that the functional abnormalities seen in muscle force measurement in COL12 KO mice originate at least partly from an underlying tendon and matrix pathology caused by the absence of collagen XII.