A novel TMPRSS3 missense mutation in a DFNB8/10 family prevents proteolytic activation of the protein.

Pathogenic mutations in TMPRSS3, which encodes a transmembrane serine protease, cause non-syndromic deafness DFNB8/10. Missense mutations map in the low density-lipoprotein receptor A (LDLRA), scavenger-receptor cysteine-rich (SRCR), and protease domains of the protein, indicating that all domains are important for its function. TMPRSS3 undergoes proteolytic cleavage and activates the ENaC sodium channel in a Xenopus oocyte model system. To assess the importance of this gene in non-syndromic childhood or congenital deafness in Turkey, we screened for mutations affected members of 25 unrelated Turkish families. The three families with the highest LOD score for linkage to chromosome 21q22.3 were shown to harbor P404L, R216L, or Q398X mutations, suggesting that mutations in TMPRSS3 are a considerable contributor to non-syndromic deafness in the Turkish population. The mutant TMPRSS3 harboring the novel R216L missense mutation within the predicted cleavage site of the protein fails to undergo proteolytic cleavage and is unable to activate ENaC, thus providing evidence that pre-cleavage of TMPRSS3 is mandatory for normal function.

Characterization and functional identification of a novel plant extradiol 4,5-dioxygenase involved in betalain pigment biosynthesis in Portulaca Grandiflora

RESUME Les bétalaïnes sont des pigments chromo-alcaloïdes violets et jaunes présents dans les plantes appartenant à l'ordre des Caryophyllales et dans les champignons des genres Amanita et Hygrocybe. Leur courte voie de biosynthèse est élucidée chimiquement depuis de nombreuses années, mais les enzymes impliquées dans cette biosynthèse chez les plantes ne sont toujours pas caractérisées. L'enzyme de la DOPA-dioxygénase d' Amanita muscaria a été identifiée (Girod et Zryd, 1991a), mais de nombreuses tentatives d'isolation d'un homologue chez les plantes ont échoué. Afin d'isoler les gènes spécifiques des bétalaïnes chez les plantes, nous avons construit des banques soustraites d'ADNc à partir d'ARN total de pétales immatures de Portulaca grandiflora (Pg) de génotypes jaunes et blancs, respectivement violets et blancs. Les clones couleur- spécifiques ont été détectés en premier par analyse Northem du RNA de pétales blancs et colorés. Les candidats positifs ont alors été soumis à une analyse de transcription au niveau des tiges colorées, vertes et des feuilles, afin d'établir leur expression spécifique. Deux ARNs messagers complets ont une expression corrélée avec l'accumulation des bétalaïnes dans les tissus. Le premier de ces clones, A.16, code pour une oxydase de l'acyl-Coenzyme A (ACX) putative, mais le domaine de liaison du FAD essentiel pour l'activité d'ACX est absent. Toutes nos tentatives pour démontrer sa fonction ont échoué. Le rôle de cette protéine dans la voie de synthèse des bétalaïnes reste inconnu. Le deuxième de ces clones spécifique aux bétalaïnes, L.6 (isolé par Zaiko, 2000), a été renommé DODA en raison de son homologie avec le domaine LigB (pfam02900) d'une 4,5-dioxygénase extradiol bactérienne. DODA a été identifié in silico comme une dioxygénase extradiol en raison de la conservation stricte, au niveau de sa séquence peptidique, des résidus catalytiques de LigB et de ceux liant le cofacteur fer. Une analyse de transfert Southem a montré que ce gène est unique dans Pg. L'expression transitoire de DODA par transformation biolistique dans des pétales blancs de Pg a produit des taches violettes ou jaunes dans des cellules transformées. Une analyse HPLC de ces taches a démontré leur identité avec les bétalaïnes présentes naturellement dans les pétales violets et jaunes de Pg, confirmant ainsi la complémentation par le gène Pg DODA de l'allèle récessif cc présent dans les pétales blancs de Pg. Des homologues de DODA (DOPA-dioxygénase) ont été identifiés dans de nombreuses espèces de plantes, y compris dans celles sans bétalaïne. L'alignement de ces homologues a permis l'identification d'un motif spécifique aux bétalaïnes à côté d'une histidine catalytique conservée. Ce motif [H-P-(S,A)-(N,D)-x-T-P] remplace le motif [H-N-L-R] conservé dans les plantes sans bétalaïne et le motif [H-N-L-x] présent dans tous les homologues bactériens et archaebactériens. Une modélisation tridimensionnelle préliminaire du site actif de Pg DODA et de son homologue dans la mousse Physcomitrella patens a montré l'importance de ce motif spécifique aux bétalaïnes pour l'accessibilité du substrat au site actif. L'analyse phylogénétique de DODA a confirmé l'évolution séparée de cette protéine chez les plantes à bétalaïnes par comparaison avec celle des plantes sans bétalaïne. Nous avons donc conclu que les bétalaïnes sont apparues par modification de l'affinité pour un substrat d'enzymes similaires à DODA, chez un ancêtre unique des Caryophyllales qui a perdu toute capacité de biosynthèse des anthocyanes. Finalement, Pg DODA n'a aucune similarité avec la protéine DODA d' Amanita muscaria, bien que celle-ci complémente aussi la pigmentation des pétales blancs de Pg. La biosynthèse des bétalaïnes est un exemple remarquable de convergence évolutive biochimique indépendante entre espèces de règnes différents. ABSTRACT Betalains are violet and yellow chromo-alkaloid pigments present in plants belonging to the order Caryophyllales and also in the fungal genera Amanita and Hygrocybe. Their short biosynthetic pathway is chemically well understood since many years, but enzymes involved in the plant pathway are still uncharacterized. The DOPA-dioxygenase from Amanita muscaria was identified (Girod and Zryd, 1991a), but numerous attempts to identify a plant homologue to the corresponding gene, failed. In order to isolate betalain-specific genes in plants, subtractive cDNA libraries were built with total RNA from white and yellow and respectively, violet immature petals from Portulaca grandiflora (Pg) genotypes. Colour-specific clones were first detected by Northern blot analysis using RNA from white and coloured petals. Positive candidates were submitted to further transcription analysis in coloured, green stems and leaves in order to assess their specific expression. Two full-length mRNAs showed a correlated expression with betalain accumulation in tissues. One of them, A.16, encodes a putative acyl-Coenzyme A oxidase (ACX), but missing the FAD binding domain essential for the ACX activity. Thus, all attempts to demonstrate its function failed. The role of this protein in the betalain biosynthesis pathway, if any, is still unknown. The second betalain-specific mRNA, L.6 (isolated by Zaiko, 2000) shows a homology with a LigB domain (pfam02900) from a bacterial extradiol 4,5-dioxygenase. It was then renamed DODA (DOPA-dioxygenase). DODA was identified in silico as a highly conserved extradiol dioxygenase due to the strict conservation of its peptidic sequence with LigB catalytic residues and iron-binding cofactor residues. Southern blot analysis showed that this gene is a single copy-gene in Pg. Transient expression of DODA protein through biolistic transformation of Pg white petals produced violet or yellow spots in individual cells. HPLC analysis of these spots showed an identity with betalain pigments present naturally in yellow and violet Pg petals, thus confirming the complementation of the recessive cc allele present in Pg white petals by Pg DODA gene. DODA homologues were identified in numerous plant species including those without betalain. Alignment of these homologues allowed the identification of a betalain-specific pattern beside a highly conserved catalytic histidine. This [H-P-(S,A)-(N,D)-x-T-P] pattern replaces a [H-N-L-R] pattern strictly conserved in non-betalain plants and a [H-N-L-x] pattern present in all bacterial and archaebacterial homologues. Preliminary three-dimensional modeling of the active site of Pg DODA and its Physcomitrella patens moss homologue revealed the importance of this betalain-specific pattern for the substrate accessibility to the DODA active site. DODA phylogenetic analysis confirmed the separate evolution of this protein in betalain-producing plants. We conclude that betalain pigments appeared in a unique ancestor of the Caryophyllales order in which anthocyanin biosynthetic pathway was impaired, by a modification of enzymes of the DODA family for substrate affinity. The Pg DODA protein has no sequence similarity with Amanita muscaria DODA, despite the fact that they both complement Pg white petals for their pigmentation. Betalain biosynthesis is an interesting example of independent biochemical evolutionary convergence between species from different kingdoms.

Corrigendum : Novel loci affecting iron homeostasis and their effects in individuals at risk for hemochromatosis

Absorption, transport and storage of iron are tightly regulated, as expected for an element, which is both essential and potentially toxic. Iron deficiency is the leading cause of anaemia, and it also compromises immune function and cognitive development. Iron overload damages the liver and other organs in hereditary hemochromatosis, and in thalassaemia patients with both transfusion and non-transfusionrelated iron accumulation. Excess iron has harmful effects in chronic liver diseases caused by excessive alcohol, obesity or viruses. There is evidence for involvement of iron in neurodegenerative diseases and in Type 2 diabetes. Variation in transferrin saturation, a biomarker of iron status, has been associated with mortality in patients with diabetes and in the general population13. All these associations between iron and either clinical disease or pathological processes make it important to understand the causes of variation in iron status. Importantly, information on genetic causes of variation can be used in Mendelian randomization studies to test whether variation in iron status is a cause or consequence of disease. We have used biomarkers of iron status (serum iron, transferrin, transferrin saturation and ferritin), which are commonly used clinically and readily measurable in thousands of individuals, and carried out a meta-analysis of human genomewide association study (GWAS) data from 11 discovery and eight replication cohorts. Our aims were to identify additional loci affecting markers of iron status in the general population and to relate the significant loci to information on gene expression to identify relevant genes. We also made an initial assessment of whether any such loci affect iron status in HFE C282Y homozygotes, who are at genetic risk of HFE-related iron overload (hereditary hemochromatosis type 1, OMIM #235200)

Genome-wide profiling of the cardiac transcriptome after myocardial infarction identifies novel heart-specific long non-coding RNAs.

AIM: Heart disease is recognized as a consequence of dysregulation of cardiac gene regulatory networks. Previously, unappreciated components of such networks are the long non-coding RNAs (lncRNAs). Their roles in the heart remain to be elucidated. Thus, this study aimed to systematically characterize the cardiac long non-coding transcriptome post-myocardial infarction and to elucidate their potential roles in cardiac homoeostasis. METHODS AND RESULTS: We annotated the mouse transcriptome after myocardial infarction via RNA sequencing and ab initio transcript reconstruction, and integrated genome-wide approaches to associate specific lncRNAs with developmental processes and physiological parameters. Expression of specific lncRNAs strongly correlated with defined parameters of cardiac dimensions and function. Using chromatin maps to infer lncRNA function, we identified many with potential roles in cardiogenesis and pathological remodelling. The vast majority was associated with active cardiac-specific enhancers. Importantly, oligonucleotide-mediated knockdown implicated novel lncRNAs in controlling expression of key regulatory proteins involved in cardiogenesis. Finally, we identified hundreds of human orthologues and demonstrate that particular candidates were differentially modulated in human heart disease. CONCLUSION: These findings reveal hundreds of novel heart-specific lncRNAs with unique regulatory and functional characteristics relevant to maladaptive remodelling, cardiac function and possibly cardiac regeneration. This new class of molecules represents potential therapeutic targets for cardiac disease. Furthermore, their exquisite correlation with cardiac physiology renders them attractive candidate biomarkers to be used in the clinic.

Genome-wide meta-analysis identifies six novel loci associated with habitual coffee consumption

Coffee, a major dietary source of caffeine, is among the most widely consumed beverages in the world and has received considerable attention regarding health risks and benefits. We conducted a genome-wide (GW) meta-analysis of predominately regular-type coffee consumption (cups per day) among up to 91 462 coffee consumers of European ancestry with top single-nucleotide polymorphisms (SNPs) followed-up in ~30 062 and 7964 coffee consumers of European and African-American ancestry, respectively. Studies from both stages were combined in a trans-ethnic meta-analysis. Confirmed loci were examined for putative functional and biological relevance. Eight loci, including six novel loci, met GW significance (log10Bayes factor (BF)>5.64) with per-allele effect sizes of 0.03-0.14 cups per day. Six are located in or near genes potentially involved in pharmacokinetics (ABCG2, AHR, POR and CYP1A2) and pharmacodynamics (BDNF and SLC6A4) of caffeine. Two map to GCKR and MLXIPL genes related to metabolic traits but lacking known roles in coffee consumption. Enhancer and promoter histone marks populate the regions of many confirmed loci and several potential regulatory SNPs are highly correlated with the lead SNP of each. SNP alleles near GCKR, MLXIPL, BDNF and CYP1A2 that were associated with higher coffee consumption have previously been associated with smoking initiation, higher adiposity and fasting insulin and glucose but lower blood pressure and favorable lipid, inflammatory and liver enzyme profiles (P<5 × 10-8).Our genetic findings among European and African-American adults reinforce the role of caffeine in mediating habitual coffee consumption and may point to molecular mechanisms underlying inter-individual variability in pharmacological and health effects of coffee.

Novel biomarkers to predict response and prognosis in localized bladder cancer.

This review summarizes recent developments in diagnostic and prognostic biomarkers for nonmuscle invasive bladder cancer (NMIBC). Although the number of new biomarkers increases continuously, none are included in practice guidelines. Most NMIBC biomarkers show a higher sensitivity than urinary cytology, but lower specificity. Some protein and chromosome markers have been approved for screening and follow-up of patients in combination with cystoscopy. The long interval required for validation, testing, and approval of the assays and the lack of standardization could explain present issues in biomarker research. To enhance the development of new biomarkers, a more structured approach is required.

Automatic Segmentation of the Eye in 3D Magnetic Resonance Imaging: A Novel Statistical Shape Model for Treatment Planning of Retinoblastoma.

PURPOSE: Proper delineation of ocular anatomy in 3-dimensional (3D) imaging is a big challenge, particularly when developing treatment plans for ocular diseases. Magnetic resonance imaging (MRI) is presently used in clinical practice for diagnosis confirmation and treatment planning for treatment of retinoblastoma in infants, where it serves as a source of information, complementary to the fundus or ultrasonographic imaging. Here we present a framework to fully automatically segment the eye anatomy for MRI based on 3D active shape models (ASM), and we validate the results and present a proof of concept to automatically segment pathological eyes. METHODS AND MATERIALS: Manual and automatic segmentation were performed in 24 images of healthy children's eyes (3.29 ± 2.15 years of age). Imaging was performed using a 3-T MRI scanner. The ASM consists of the lens, the vitreous humor, the sclera, and the cornea. The model was fitted by first automatically detecting the position of the eye center, the lens, and the optic nerve, and then aligning the model and fitting it to the patient. We validated our segmentation method by using a leave-one-out cross-validation. The segmentation results were evaluated by measuring the overlap, using the Dice similarity coefficient (DSC) and the mean distance error. RESULTS: We obtained a DSC of 94.90 ± 2.12% for the sclera and the cornea, 94.72 ± 1.89% for the vitreous humor, and 85.16 ± 4.91% for the lens. The mean distance error was 0.26 ± 0.09 mm. The entire process took 14 seconds on average per eye. CONCLUSION: We provide a reliable and accurate tool that enables clinicians to automatically segment the sclera, the cornea, the vitreous humor, and the lens, using MRI. We additionally present a proof of concept for fully automatically segmenting eye pathology. This tool reduces the time needed for eye shape delineation and thus can help clinicians when planning eye treatment and confirming the extent of the tumor.

Novel Genome-Editing Tools to Model and Correct Primary Immunodeficiencies.

Severe combined immunodeficiency (SCID) and other severe non-SCID primary immunodeficiencies (non-SCID PID) can be treated by allogeneic hematopoietic stem cell (HSC) transplantation, but when histocompatibility leukocyte antigen-matched donors are lacking, this can be a high-risk procedure. Correcting the patient's own HSCs with gene therapy offers an attractive alternative. Gene therapies currently being used in clinical settings insert a functional copy of the entire gene by means of a viral vector. With this treatment, severe complications may result due to integration within oncogenes. A promising alternative is the use of endonucleases such as ZFNs, TALENs, and CRISPR/Cas9 to introduce a double-stranded break in the DNA and thus induce homology-directed repair. With these genome-editing tools a correct copy can be inserted in a precisely targeted "safe harbor." They can also be used to correct pathogenic mutations in situ and to develop cellular or animal models needed to study the pathogenic effects of specific genetic defects found in immunodeficient patients. This review discusses the advantages and disadvantages of these endonucleases in gene correction and modeling with an emphasis on CRISPR/Cas9, which offers the most promise due to its efficacy and versatility.

Novel micropatterns mechanically control fibrotic reactions at the surface of silicone implants.

Over the past decade, various implantable devices have been developed to treat diseases that were previously difficult to manage such diabetes, chronic pain, and neurodegenerative disorders. However, translation of these novel technologies into clinical practice is often difficult because fibrotic encapsulation and/or rejection impairs device function after body implantation. Ideally, cells of the host tissue should perceive the surface of the implant being similar to the normal extracellular matrix. Here, we developed an innovative approach to provide implant surfaces with adhesive protein micropatterns. The patterns were designed to promote adhesion of fibroblasts and macrophages by simultaneously suppressing fibrogenic activation of both cell types. In a rat model, subcutaneously implanted silicone pads provided with the novel micropatterns caused 6-fold lower formation of inflammatory giant cells compared with clinical grade, uncoated, or collagen-coated silicone implants. We further show that micropatterning of implants resulted in 2-3-fold reduced numbers of pro-fibrotic myofibroblast by inhibiting their mechanical activation. Our novel approach allows controlled cell attachment to implant surfaces, representing a critical advance for enhanced biointegration of implantable medical devices.

RNA interference screening identifies a novel role for PCTK1/CDK16 in medulloblastoma with c-Myc amplification.

Medulloblastoma (MB) is the most common malignant brain tumor in children and is associated with a poor outcome. cMYC amplification characterizes a subgroup of MB with very poor prognosis. However, there exist so far no targeted therapies for the subgroup of MB with cMYC amplification. Here we used kinome-wide RNA interference screening to identify novel kinases that may be targeted to inhibit the proliferation of c-Myc-overexpressing MB. The RNAi screen identified a set of 5 genes that could be targeted to selectively impair the proliferation of c-Myc-overexpressing MB cell lines: AKAP12 (A-kinase anchor protein), CSNK1α1 (casein kinase 1, alpha 1), EPHA7 (EPH receptor A7) and PCTK1 (PCTAIRE protein kinase 1). When using RNAi and a pharmacological inhibitor selective for PCTK1, we could show that this kinase plays a crucial role in the proliferation of MB cell lines and the activation of the mammalian target of rapamycin (mTOR) pathway. In addition, pharmacological PCTK1 inhibition reduced the expression levels of c-Myc. Finally, targeting PCTK1 selectively impaired the tumor growth of c-Myc-overexpressing MB cells in vivo. Together our data uncover a novel and crucial role for PCTK1 in the proliferation and survival of MB characterized by cMYC amplification.

Homozygosity mapping reveals novel and known mutations in Pakistani families with inherited retinal dystrophies.

Inherited retinal dystrophies are phenotypically and genetically heterogeneous. This extensive heterogeneity poses a challenge when performing molecular diagnosis of patients, especially in developing countries. In this study, we applied homozygosity mapping as a tool to reduce the complexity given by genetic heterogeneity and identify disease-causing variants in consanguineous Pakistani pedigrees. DNA samples from eight families with autosomal recessive retinal dystrophies were subjected to genome wide homozygosity mapping (seven by SNP arrays and one by STR markers) and genes comprised within the detected homozygous regions were analyzed by Sanger sequencing. All families displayed consistent autozygous genomic regions. Sequence analysis of candidate genes identified four previously-reported mutations in CNGB3, CNGA3, RHO, and PDE6A, as well as three novel mutations: c.2656C > T (p.L886F) in RPGRIP1, c.991G > C (p.G331R) in CNGA3, and c.413-1G > A (IVS6-1G > A) in CNGB1. This latter mutation impacted pre-mRNA splicing of CNGB1 by creating a -1 frameshift leading to a premature termination codon. In addition to better delineating the genetic landscape of inherited retinal dystrophies in Pakistan, our data confirm that combining homozygosity mapping and candidate gene sequencing is a powerful approach for mutation identification in populations where consanguineous unions are common.

Rhumatologie. Rhumatisme psoriasique [Update in psoriatic arthritis treatment].

Psoriatic arthritis is a chronic inflammatory disease. It affects up to 40% of patients suffe- ring from skin psoriasis. Joint involvement is relatively heterogeneous. Some clinical manifestations are similar to those of rheumatoid arthritis, others are close to spondylarthritis manifestations and are therefore considered as part of this entity. Treatment depends on initial presentation (peripheral or axial) but often begins with non-steroidal anti-inflammatory drugs and methotrexate, followed by anti-TNFalpha if needed. New therapeutic op- tions are available or under evaluation, parti- cularly targeting cytokines involved in psoriatic arthritis (IL-12/IL-23 and IL-17).

Review of quantitative phase-digital holographic microscopy: promising novel imaging technique to resolve neuronal network activity and identify cellular biomarkers of psychiatric disorders.

Quantitative phase microscopy (QPM) has recently emerged as a new powerful quantitative imaging technique well suited to noninvasively explore a transparent specimen with a nanometric axial sensitivity. In this review, we expose the recent developments of quantitative phase-digital holographic microscopy (QP-DHM). Quantitative phase-digital holographic microscopy (QP-DHM) represents an important and efficient quantitative phase method to explore cell structure and dynamics. In a second part, the most relevant QPM applications in the field of cell biology are summarized. A particular emphasis is placed on the original biological information, which can be derived from the quantitative phase signal. In a third part, recent applications obtained, with QP-DHM in the field of cellular neuroscience, namely the possibility to optically resolve neuronal network activity and spine dynamics, are presented. Furthermore, potential applications of QPM related to psychiatry through the identification of new and original cell biomarkers that, when combined with a range of other biomarkers, could significantly contribute to the determination of high risk developmental trajectories for psychiatric disorders, are discussed.