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Contains fulltext : 125691.pdf (Publisher’s version ) (Closed access) OBJECTIVE: Retinitis pigmentosa (RP) is a clinically and genetically heterogeneous disorder characterized by night blindness and peripheral vision loss, and in many cases leads to blindness. Despite extensive knowledge about genes involved in the pathogenesis of RP, the genetic cause remains elusive in many patients. In this study, we aimed to identify novel genes that are involved in the cause of RP. DESIGN: We present a case series with mutations in the mevalonate kinase (MVK) gene. PARTICIPANTS: A total of 769 patients with nonsyndromic RP and 174 Dutch control individuals participated in this study. METHODS: Exome sequencing analysis was performed in a proband of Dutch origin who was initially diagnosed with nonsyndromic autosomal recessive RP. Mutations in MVK were identified and subsequently tested for segregation within the patient's family and screened in a large cohort of patients with genetically unsolved RP. Patients with mutations underwent extensive clinical reexamination. MAIN OUTCOME MEASURES: Digital fundus photography, spectral-domain optical coherence tomography (OCT), and fundus autofluorescence analysis were performed in patients with MVK mutations. Mevalonate kinase (MK) enzyme activity was analyzed in cultured lymphoblastoid cells, and mevalonic acid levels were measured in urine samples. RESULTS: Exome variant filtering and prioritization led to the identification of compound heterozygous mutations in MVK (p.I268T and p.A334T) in the proband and her affected brother. Screening of our nonsyndromic RP patient cohort revealed an additional individual who was homozygous for the p.A334T alteration. Clinical reevaluation of all 3 patients showed a classic form of RP with variable extraocular symptoms, such as history of recurrent childhood febrile crises in 2 patients, mild ataxia in 1, and renal failure in 1. All 3 affected individuals showed a significantly decreased MK activity and highly elevated levels of urinary mevalonic acid. CONCLUSIONS: Although the MK activity in cells and mevalonic acid concentrations in urine are strongly aberrant and comparable to that in patients with systemic mevalonate kinase deficiency (MKD), only mild clinical symptoms related to this syndrome were observed in our patients. In the current article, we add another phenotype to the spectrum of diverging disorders associated with mutations in MVK. FINANCIAL DISCLOSURE(S): The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Contains fulltext : 125451.pdf (Publisher’s version ) (Closed access) Familial hemiplegic migraine (FHM) is a monogenic variant of migraine with aura. One of the three known causative genes, ATP1A2, which encodes the alpha2 isoform of Na,K-ATPase, causes FHM type 2 (FHM2). Over 50 FHM2 mutations have been reported, but most have not been characterized functionally. Here we study the molecular mechanism of Na,K-ATPase alpha2 missense mutations. Mutants E700K and P786L inactivate or strongly reduce enzyme activity. Glutamic acid 700 is located in the phosphorylation (P) domain and the mutation most likely disrupts the salt bridge with Lysine 35, thereby destabilizing the interaction with the actuator (A) domain. Mutants G900R and E902K are present in the extracellular loop at the interface of the alpha and beta subunit. Both mutants likely hamper the interaction between these subunits and thereby decrease enzyme activity. Mutants E174K, R548C and R548H reduce the Na(+) and increase the K(+) affinity. Glutamic acid 174 is present in the A domain and might form a salt bridge with Lysine 432 in the nucleotide binding (N) domain, whereas Arginine 548, which is located in the N domain, forms a salt bridge with Glutamine 219 in the A domain. In the catalytic cycle, the interactions of the A and N domains affect the K(+) and Na(+) affinities, as observed with these mutants. Functional consequences were not observed for ATP1A2 mutations found in two sporadic hemiplegic migraine cases (Y9N and R879Q) and in migraine without aura (R51H and C702Y).

Item does not contain fulltext Vitelliform macular dystrophies (VMD) are inherited retinal dystrophies characterized by yellow, round deposits visible upon fundus examination and encountered in individuals with juvenile Best macular dystrophy (BMD) or adult-onset vitelliform macular dystrophy (AVMD). Although many BMD and some AVMD cases harbor mutations in BEST1 or PRPH2, the underlying genetic cause remains unknown for many affected individuals. In a large family with autosomal-dominant VMD, gene mapping and whole-exome sequencing led to the identification of a c.713T>G (p.Leu238Arg) IMPG1 mutation, which was subsequently found in two other families with autosomal-dominant VMD and the same phenotype. IMPG1 encodes the SPACR protein, a component of the rod and cone photoreceptor extracellular matrix domains. Structural modeling indicates that the p.Leu238Arg substitution destabilizes the conserved SEA1 domain of SPACR. Screening of 144 probands who had various forms of macular dystrophy revealed three other IMPG1 mutations. Two individuals from one family affected by autosomal-recessive VMD were homozygous for the splice-site mutation c.807+1G>T, and two from another family were compound heterozygous for the mutations c.461T>C (p.Leu154Pro) and c.1519C>T (p.Arg507( *)). Most cases had a normal or moderately decreased electrooculogram Arden ratio. We conclude that IMPG1 mutations cause both autosomal-dominant and -recessive forms of VMD, thus indicating that impairment of the interphotoreceptor matrix might be a general cause of VMD.

Item does not contain fulltext The gene products of the five-membered PRS gene family in Saccharomyces cerevisiae have been shown to exist as three minimal functional entities, Prs1/Prs3, Prs2/Prs5, and Prs4/Prs5, each capable of supporting cell viability. The Prs1/Prs3 heterodimer can be regarded as the most important because its loss causes temperature sensitivity. It has been shown that the GFP signal generated by an integrated GFP-Prs1 construct is lost in the absence of Prs3. In addition to interacting with Prs3, Prs1 also interacts with Slt2, the MAPK of the cell wall integrity (CWI) pathway. Lack of the nonhomologous region (NHR1-1) located centrally in Prs1 abolished the temperature-induced increase in Rlm1 expression. Furthermore, in vitro point mutations generated in PRS1 corresponding to missense mutations associated with human neuropathies or in the divalent cation and/or 5-phosphoribosyl-1(alpha)-pyrophosphate binding sites also display increased Rlm1 expression at 30 degrees C and 37 degrees C and most give rise to caffeine sensitivity. Human PRPS1 cDNA cannot rescue the synthetic lethality of a prs1Delta prs5Delta strain because it lacks sequences corresponding to NHR1-1 of yeast Prs1. The correlation between caffeine sensitivity and increased basal expression of Rlm1 in the altered versions of PRS1 can be extended to their inability to rescue the synthetic lethality of a prs1Delta prs5Delta strain implying that impaired CWI may contribute to the observed loss of viability.

Item does not contain fulltext The majority of the genetic causes of autosomal-recessive (ar) cone-rod dystrophy (CRD) are currently unknown. A combined approach of homozygosity mapping and exome sequencing revealed a homozygous nonsense mutation (c.565C>T [p.Glu189*]) in RAB28 in a German family with three siblings with arCRD. Another homozygous nonsense mutation (c.409C>T [p.Arg137*]) was identified in a family of Moroccan Jewish descent with two siblings affected by arCRD. All five affected individuals presented with hyperpigmentation in the macula, progressive loss of the visual acuity, atrophy of the retinal pigment epithelium, and severely reduced cone and rod responses on the electroretinogram. RAB28 encodes a member of the Rab subfamily of the RAS-related small GTPases. Alternative RNA splicing yields three predicted protein isoforms with alternative C-termini, which are all truncated by the nonsense mutations identified in the arCRD families in this report. Opposed to other Rab GTPases that are generally geranylgeranylated, RAB28 is predicted to be farnesylated. Staining of rat retina showed localization of RAB28 to the basal body and the ciliary rootlet of the photoreceptors. Analogous to the function of other RAB family members, RAB28 might be involved in ciliary transport in photoreceptor cells. This study reveals a crucial role for RAB28 in photoreceptor function and suggests that mutations in other Rab proteins may also be associated with retinal dystrophies.

Item does not contain fulltext Inherited retinal diseases display a very high degree of clinical and genetic heterogeneity, which poses challenges in identifying the underlying defects in known genes and in identifying novel retinal disease genes. Here, we outline the state-of-the-art techniques to find the causative DNA variants, with special attention for next-generation sequencing which can combine molecular diagnostics and retinal disease gene identification.

Contains fulltext : 118015.pdf (Publisher’s version ) (Open Access) PURPOSE: The association of non-synonymous substitution polymorphism rs1801282 (c.34C>G, p.Pro12Ala) in exon 4 of the peroxisome proliferator activated receptor gamma gene with diabetic retinopathy (DR) has been reported inconsistently. Therefore, the purpose of the present study was to understand the population-specific role of the Pro12Ala polymorphism in DR susceptibility in Pakistani subjects. METHODS: A total of 180 subjects with DR, 193 subjects with type 2 diabetes mellitus (T2DM) with no diabetic retinopathy, and 200 healthy normoglycemic non-retinopathic Pakistani individuals were genotyped for the rs1801282 (c.34C>G) polymorphism using polymerase chain reaction-restriction fragment length polymorphism. RESULTS: We found the individuals with T2DM carrying 12Ala were at a reduced risk of developing DR (odds ratio [OR]=0.53; 95% confidence interval [CI]=0.33-0.87). Upon stratified analysis regarding disease severity, we observed this protective effect was confined to proliferative DR (OR=0.4; 95% CI=0.2-0.8) with non-significant effects on the susceptibility of non-proliferative DR (OR=0.67; 95% CI=0.37-1.19). CONCLUSIONS: We report a protective role of the 12Ala polymorphism against proliferative DR in individuals with T2DM in Pakistan.

Familial exudative vitreoretinopathy (FEVR) is a genetically heterogeneous disorder characterized by abnormal vascularization of the peripheral retina, which can result in retinal detachment and severe visual impairment. In a large Dutch FEVR family, we performed linkage analysis, exome sequencing, and segregation analysis of DNA variants. We identified putative disease-causing DNA variants in proline-alanine-rich ste20-related kinase (c.791dup; p.Ser265ValfsX64) and zinc finger protein 408 ( ZNF408 ) (c.1363C>T; p.His455Tyr), the latter of which was also present in an additional Dutch FEVR family that subsequently appeared to share a common ancestor with the original family. Sequence analysis of ZNF408 in 132 additional individuals with FEVR revealed another potentially pathogenic missense variant, p.Ser126Asn, in a Japanese family. Immunolocalization studies in COS-1 cells transfected with constructs encoding the WT and mutant ZNF408 proteins, revealed that the WT and the p.Ser126Asn mutant protein show complete nuclear localization, whereas the p.His455Tyr mutant protein was localized almost exclusively in the cytoplasm. Moreover, in a cotransfection assay, the p.His455Tyr mutant protein retains the WT ZNF408 protein in the cytoplasm, suggesting that this mutation acts in a dominant-negative fashion. Finally, morpholino-induced knockdown of znf408 in zebrafish revealed defects in developing retinal and trunk vasculature, that could be rescued by coinjection of RNA encoding human WT ZNF408 but not p.His455Tyr mutant ZNF408. Together, our data strongly suggest that mutant ZNF408 results in abnormal retinal vasculogenesis in humans and is associated with FEVR.

Contains fulltext : 118540.pdf (Publisher’s version ) (Closed access)

Contains fulltext : 117375.pdf (Publisher’s version ) (Open Access) An increasing number of genes involved in chromatin structure and epigenetic regulation has been implicated in a variety of developmental disorders, often including intellectual disability. By trio exome sequencing and subsequent mutational screening we now identified two de novo frameshift mutations and one de novo missense mutation in CTCF in individuals with intellectual disability, microcephaly, and growth retardation. Furthermore, an individual with a larger deletion including CTCF was identified. CTCF (CCCTC-binding factor) is one of the most important chromatin organizers in vertebrates and is involved in various chromatin regulation processes such as higher order of chromatin organization, enhancer function, and maintenance of three-dimensional chromatin structure. Transcriptome analyses in all three individuals with point mutations revealed deregulation of genes involved in signal transduction and emphasized the role of CTCF in enhancer-driven expression of genes. Our findings indicate that haploinsufficiency of CTCF affects genomic interaction of enhancers and their regulated gene promoters that drive developmental processes and cognition.

Contains fulltext : 117693.pdf (Publisher’s version ) (Closed access) The TcPOT1.1 gene from Trypanosoma cruzi encodes a high affinity putrescine-cadaverine transporter belonging to the APC (amino acid/polyamine/organocation) transporter superfamily. No experimental three-dimensional structure exists for any eukaryotic member of the APC family, and thus the structural determinants critical for function of these permeases are unknown. To elucidate the key residues involved in putrescine translocation and recognition by this APC family member, a homology model of TcPOT1.1 was constructed on the basis of the atomic co-ordinates of the Escherichia coli AdiC arginine/agmatine antiporter crystal structure. The TcPOT1.1 homology model consisted of 12 transmembrane helices with the first ten helices organized in two V-shaped antiparallel domains with discontinuities in the helical structures of transmembrane spans 1 and 6. The model suggests that Trp241 and a Glu247-Arg403 salt bridge participate in a gating system and that Asn245, Tyr148 and Tyr400 contribute to the putrescine-binding pocket. To test the validity of the model, 26 site-directed mutants were created and tested for their ability to transport putrescine and to localize to the parasite cell surface. These results support the robustness of the TcPOT1.1 homology model and reveal the importance of specific aromatic residues in the TcPOT1.1 putrescine-binding pocket.

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Item does not contain fulltext Consensus engineering, which is replacing amino acids by the most frequently occurring one at their positions in a multiple sequence alignment (MSA), is a known strategy to increase the stability of a protein. The application of this concept to the entire sequence of an enzyme, however, has been tried only a few times mainly because of the problems determining the consensus in highly variable regions. We show that this problem can be solved by replacing such problematic regions by the corresponding sequence of the natural homologue closest to the consensus. When one or a few sub-families are overrepresented in the MSA the consensus sequence is a biased representation of the sequence space. We examine the influence of this bias by constructing three consensus sequences using different MSAs of sucrose phosphorylase (SP). Each consensus enzyme contained about 70 mutations compared to its closest natural homologue and folded correctly and displayed activity on sucrose. Correlation analysis revealed that the family's co-evolution network was kept intact, which is one of the main advantages of full-length consensus design. The consensus enzymes displayed an "average" thermostability, that is, one that is higher than some but not all known representatives. We cautiously present practical rules for the design of consensus sequences, but warn that the measure of success depends on which natural enzyme is used as point of comparison. Biotechnol. Bioeng. 2013;110: 2563-2572. (c) 2013 Wiley Periodicals, Inc.

Item does not contain fulltext SETTINGS: Private pharmacies in Hanoi, Viet Nam. OBJECTIVES: To explore the response of health care providers (HCPs) in private pharmacies to suspected tuberculosis (TB) patients. METHODS: A simulated patient method combined with an interview in 128 randomly selected private pharmacies and 10 private pharmacies near TB hospitals. RESULTS: In the simulated patient method and interview, respectively 59 (46%) and 70 (55%) of HCPs referred the TB suspect to general health care. Only 11 (9%) referred the simulated patient to a TB care facility. Fifty-two (42%) of the HCPs identified suspected TB from a fictitious case described on paper; 34 (27%) were aware that free treatment was provided under the National Tuberculosis Programme (NTP). Knowledge about free NTP treatment predicted a higher rate of direct referrals to TB facilities (OR 5.80, 95%CI 1.88-19.62) and greater ability to identify suspected TB from a fictitious case on paper (OR 5.14, 95%CI 2.36-11.73). Pharmacies with Good Pharmacy Practice (GPP) certification were less likely to refer simulated patients to TB facilities than non-GPP pharmacies (OR 0.10, 95%CI

Contains fulltext : 118008.pdf (Publisher’s version ) (Open Access) Ankyloblepharon, ectodermal defects, cleft lip/palate (AEC) syndrome is a rare autosomal dominant disorder caused by mutations in the p63 gene, essential for embryonic development of stratified epithelia. The most severe cutaneous manifestation of this disorder is the long-lasting skin fragility associated with severe skin erosions after birth. Using a knock-in mouse model for AEC syndrome, we found that skin fragility was associated with microscopic blistering between the basal and suprabasal compartments of the epidermis and reduced desmosomal contacts. Expression of desmosomal cadherins and desmoplakin was strongly reduced in AEC mutant keratinocytes and in newborn epidermis. A similar impairment in desmosome gene expression was observed in human keratinocytes isolated from AEC patients, in p63-depleted keratinocytes and in p63 null embryonic skin, indicating that p63 mutations causative of AEC syndrome have a dominant-negative effect on the wild-type p63 protein. Among the desmosomal components, desmocollin 3, desmoplakin and desmoglein 1 were the most significantly reduced by mutant p63 both at the RNA and protein levels. Chromatin immunoprecipitation experiments and transactivation assays revealed that p63 controls these genes at the transcriptional level. Consistent with reduced desmosome function, AEC mutant and p63-deficient keratinocytes had an impaired ability to withstand mechanical stress, which was alleviated by epidermal growth factor receptor inhibitors known to stabilize desmosomes. Our study reveals that p63 is a crucial regulator of a subset of desmosomal genes and that this function is impaired in AEC syndrome. Reduced mechanical strength resulting from p63 mutations can be alleviated pharmacologically by increasing desmosome adhesion with possible therapeutic implications.

Item does not contain fulltext Immunological memory in vertebrates is often exclusively attributed to T and B cell function. Recently it was proposed that the enhanced and sustained innate immune responses following initial infectious exposure may also afford protection against reinfection. Testing this concept of "trained immunity," we show that mice lacking functional T and B lymphocytes are protected against reinfection with Candida albicans in a monocyte-dependent manner. C. albicans and fungal cell wall beta-glucans induced functional reprogramming of monocytes, leading to enhanced cytokine production in vivo and in vitro. The training required the beta-glucan receptor dectin-1 and the noncanonical Raf-1 pathway. Monocyte training by beta-glucans was associated with stable changes in histone trimethylation at H3K4, which suggests the involvement of epigenetic mechanisms in this phenomenon. The functional reprogramming of monocytes, reminiscent of similar NK cell properties, supports the concept of "trained immunity" and may be employed for the design of improved vaccination strategies.

Nephronophthisis-related ciliopathies (NPHP-RC) are degenerative recessive diseases that affect kidney, retina and brain. Genetic defects in NPHP gene products that localize to cilia and centrosomes defined them as ‘ciliopathies’. However, disease mechanisms remain poorly understood. Here we identify by whole exome resequencing, mutations of MRE11, ZNF423, and CEP164 as causing NPHP-RC. All three genes function within the DNA damage response (DDR) pathway, hitherto not implicated in ciliopathies. We demonstrate that, upon induced DNA damage, the NPHP-RC proteins ZNF423, CEP164 and NPHP10 colocalize to nuclear foci positive for TIP60, known to activate ATM at sites of DNA damage. We show that knockdown of CEP164 or ZNF423 causes sensitivity to DNA damaging agents, and that cep164 knockdown in zebrafish results in dysregulated DDR and an NPHP-RC phenotype. We identify TTBK2, CCDC92, NPHP3 and DVL3 as novel CEP164 interaction partners. Our findings link degenerative diseases of kidney and retina, disorders of increasing prevalence, to mechanisms of DDR.

Contains fulltext : 103842.pdf (Publisher’s version ) (Closed access) The adenosine A1 receptor is a member of the large membrane protein family that signals through G proteins, the G protein-coupled receptors (GPCRs). GPCRs consist of seven transmembrane domains connected by three intracellular and three extracellular loops. Their N-terminus is extracellular, the C-terminal tail is in the cytoplasm. The transmembrane domains in receptor subfamilies that bind the same endogenous ligand, such as dopamine or adenosine, tend to be highly similar. In contrast, the loop regions can vary greatly, both in sequence and in length, and the role these loops have in the activation mechanism of the receptors remains unclear. Here, we investigated the activating role of the second and third extracellular loop of the human adenosine A1 receptor. By means of an (Ala)3 mutagenic scan in which consecutive sets of three amino acids were mutated into alanine residues in EL2 and a classical alanine scan in EL3, we revealed a strong regulatory role for the second extracellular loop (EL2) of the human adenosine A1 receptor. Besides many residues in the second and the third extracellular loops important for adenosine A1 receptor activation, we also identified two residues in EL2, a tryptophan and a glutamate, that affect the influence of the allosteric modulator PD81,723. These results, combined with a comparison of the different receptor loop regions, provide insight in the activation mechanism of this typical class A GPCR and further emphasize the unique pharmacological profile the loops can provide to individual receptors, even within subfamilies of GPCRs.

Item does not contain fulltext Nemaline myopathy and myofibrillar myopathy are heterogeneous myopathies that both comprise early-onset forms. We present two sisters from a consanguineous Iraqi Kurdish family with predominant axial and limb girdle weakness. Muscle biopsies showed features of both nemaline myopathy and myofibrillar myopathy. We performed homozygosity mapping in both siblings using an Affymetrix 250K Nspl SNP array. One of the overlapping homozygous regions harbored the gene CFL2. Because a mutation in CFL2 was identified in a family with nemaline myopathy, we performed sequence analysis of the gene and a novel homozygous missense mutation in exon 2 (c.19G>A, p.Val7Met) of CFL2 was identified in both siblings. CFL2 encodes the protein cofilin-2, which plays an important role in regulation of sarcomeric actin filaments. To our knowledge, this is the second family in which a mutation in CFL2 causes an autosomal recessive form of congenital myopathy with features of both nemaline and myofibrillar myopathy. Given the clinical variability and the multitude of histological features of congenital myopathies, CFL2 sequence analysis should be considered in patients presenting with an autosomal recessive form of congenital myopathy. 01 juli 2012

Item does not contain fulltext Mutations in COCH have been associated with autosomal dominant nonsyndromic hearing loss (DFNA9) and are frequently accompanied by vestibular hypofunction. Here, we report identification of a novel missense mutation, p.F527C, located in the vWFA2 domain in members of a Korean family with late-onset and progressive hearing loss. To assess the molecular characteristics of this cochlin mutant, we constructed both wild-type and mutant cochlin constructs and transfected these into mammalian cell lines. Results of immunocytochemistry analysis demonstrated localization of the cochlin mutant in the endoplasmic reticulum/Golgi complex, whereas western blot analyses of cell lysates revealed that the mutant cochlin tends to form covalent complexes that are retained in the cell. Biochemical analyses of recombinant vWFA2 domain of cochlin carrying the p.F527C mutation revealed that the mutation increases propensity of the protein to form covalent disulfide-bonded dimers and affects the structural stability but not the collagen-affinity of the vWFA2 domain. We suggest that the instability of mutant cochlin is the major driving force for cochlin aggregation in the inner ear in DFNA9 patients carrying the COCH p.F527C mutation.

Contains fulltext : 110974.pdf (Publisher’s version ) (Open Access) PURPOSE: Genetic studies were performed to identify the causative mutation in a 15-year-old girl diagnosed with congenital stationary night blindness (CSNB) presenting Mizuo-Nakamura phenomenon, a typical Oguchi disease symptom. The patient also had dural sinus thrombosis (DST), thrombocytopenia, and systemic lupus erythematosus (SLE). METHODS: Mutation analysis was done by sequencing two candidate genes, S-antigen (SAG; arrestin 1), associated with Oguchi type 1, and rhodopsin kinase (GRK1), associated with Oguchi type 2. In addition, the C677T variation in the methylenetetrahydrofolate reductase (MTHFR) gene was also screened in the family, to determine its probable association with hyperhomocysteinemia in the patient. RESULTS: Sequencing of the SAG and GRK1 resulted in identifying a novel homozygous nonsense mutation (c.916G>T; p.Glu306*) in SAG, which in unaffected siblings either was present in a heterozygous state or absent. The C677T heterozygous allele in the MTHFR gene was found to be associated with hyperhomocysteinemia in the patient and other family members. CONCLUSIONS: This is the first report of Oguchi type 1 in a Pakistani patient due to a nonsense mutation (c.916G>T; p.Glu306*) in SAG. The neurologic and hematological abnormalities likely are not associated with the SAG variant.

Item does not contain fulltext Mercury pollution is a major environmental problem that arises as a result of natural processes as well as from anthropogenic sources. In response to toxic mercury compounds, microbes have developed astonishing array of resistance systems to detoxify them. To address this challenge, this study was aimed in screening bacterial isolates for their tolerance against varied concentrations of phenylmercuric acetate. Mercury transformation by bacteria being sensitive to factors such as available carbon source, etc. that affect mer-mediated transformation, screened mercury tolerant bacteria were also studied for their tolerance to different antimicrobials and carbon sources, followed by identification using biochemical as well as 16S rRNA approach. Following identification, gene encoding organomercurial lyase catalyzing protonolytic cleavage of C-Hg bond of organic mercury was amplified using gene specific primers, cloned in pGEMT((R)) easy vector and sequenced. Microbe-based approach using organomercurial lyase encoded by merB gene being potentially economic, provides foundation to facilitate genetic manipulation of this environmentally important enzyme to remove high concentrations of obstinate mercury using holistic, multifaceted approach for use in bioremediation through generation of transgenics or as catalyst for use in bioreactors.

Item does not contain fulltext Bone morphogenic protein-2 (BMP-2) is a well-known growth factor that can improve the biological performance of bone substitute materials. BMP-2 produced via bacterial expression systems are non-glycosylated (ng) whereas native and recombinant equivalents produced in mammalian cell expression systems are glycosylated (g) proteins. ngBMP-2 is less soluble, resulting in lower BMP-2 release from carriers as used as bone substitute materials. This seems promising for reducing the amount of included growth factor in bone substitute materials. Hence, it was hypothesized that ngBMP-2 would induce formation of the same amount of bone at an ectopic site at lower dosage as gBMP-2. To that end, gBMP-2 and ngBMP-2 were firstly in vitro comparatively evaluated for biological activity and release from a calcium phosphate (CaP) based bone substitute material. Thereafter, an ectopic implantation model in rats was used, in which gBMP-2 and ngBMP2 were loaded in various dosages (2-20 mug/implant) on the CaP-based bone substitute material and implanted for 4 and 12 weeks. The results revealed that both the in vitro biological activity of and the in vitro release of ngBMP-2 are lower compared to gBMP2. Upon ectopic implantation, however, ngBMP-2 loaded implants induced more bone formation at lower concentrations from 4-weeks onward compared to gBMP-2 equivalents, indicating the value of ngBMP-2 as a potential alternative for mammalian produced recombinant BMP-2 for bone regenerative therapies.

Item does not contain fulltext Coordinates describing the chemical structures of small molecules that are potential ligands for pharmaceutical targets are used at many stages of the drug design process. The coordinates of the vast majority of ligands can be obtained from either publicly accessible or commercial databases. However, interesting ligands sometimes are only available from the scientific literature, in which case their coordinates need to be reconstructed manually--a process that consists of a series of time-consuming steps. We present a Web server that helps reconstruct the three-dimensional (3D) coordinates of ligands for which a two-dimensional (2D) picture is available in a PDF file. The software, called AsteriX, analyses every picture contained in the PDF file and attempts to determine automatically whether or not it contains ligands. Areas in pictures that may contain molecular structures are processed to extract connectivity and atom type information that allow coordinates to be subsequently reconstructed. The AsteriX Web server was tested on a series of articles containing a large diversity in graphical representations. In total, 88% of 3249 ligand structures present in the test set were identified as chemical diagrams. Of these, about half were interpreted correctly as 3D structures, and a further one-third required only minor manual corrections. It is principally impossible to always correctly reconstruct 3D coordinates from pictures because there are many different protocols for drawing a 2D image of a ligand, but more importantly a wide variety of semantic annotations are possible. The AsteriX Web server therefore includes facilities that allow the users to augment partial or partially correct 3D reconstructions. All 3D reconstructions are submitted, checked, and corrected by the users domain at the server and are freely available for everybody. The coordinates of the reconstructed ligands are made available in a series of formats commonly used in drug design research. The AsteriX Web server is freely available at http://swift.cmbi.ru.nl/bitmapb/.

Contains fulltext : 108772.pdf (Publisher’s version ) (Open Access) Walker-Warburg syndrome (WWS) is an autosomal recessive multisystem disorder characterized by complex eye and brain abnormalities with congenital muscular dystrophy (CMD) and aberrant a-dystroglycan glycosylation. Here we report mutations in the ISPD gene (encoding isoprenoid synthase domain containing) as the second most common cause of WWS. Bacterial IspD is a nucleotidyl transferase belonging to a large glycosyltransferase family, but the role of the orthologous protein in chordates is obscure to date, as this phylum does not have the corresponding non-mevalonate isoprenoid biosynthesis pathway. Knockdown of ispd in zebrafish recapitulates the human WWS phenotype with hydrocephalus, reduced eye size, muscle degeneration and hypoglycosylated a-dystroglycan. These results implicate ISPD in a-dystroglycan glycosylation in maintaining sarcolemma integrity in vertebrates.

Contains fulltext : 110709.pdf (Publisher’s version ) (Closed access) Three-component coupling, with a pair: The combination of Cu(II) and Ti(IV) catalyzes the first three-component coupling of unactivated ketones with a diverse range of amines and terminal alkynes. Tetrasubstituted propargylamines are formed under green, solvent-free conditions. This dual metal system overcomes the barrier to ketimine formation and subsequent attack, opening a new path to multicomponent reactions of ketone electrophiles.

Item does not contain fulltext Homozygosity mapping and exome sequencing have accelerated the discovery of gene mutations and modifier alleles implicated in inherited retinal degeneration in humans. To date, 158 genes have been found to be mutated in individuals with retinal dystrophies. Approximately one-third of the gene defects underlying retinal degeneration affect the structure and/or function of the 'connecting cilium' in photoreceptors. This structure corresponds to the transition zone of a prototypic cilium, a region with increasing relevance for ciliary homeostasis. The connecting cilium connects the inner and outer segments of the photoreceptor, mediating bi-directional transport of phototransducing proteins required for vision. In fact, the outer segment, connecting cilium and associated basal body, forms a highly specialized sensory cilium, fully dedicated to photoreception and subsequent signal transduction to the brain. At least 21 genes that encode ciliary proteins are implicated in non-syndromic retinal dystrophies such as cone dystrophy, cone-rod dystrophy, Leber congenital amaurosis (LCA), macular degeneration or retinitis pigmentosa (RP). The generation and characterization of vertebrate retinal ciliopathy animal models have revealed insights into the molecular disease mechanism which are indispensable for the development and evaluation of therapeutic strategies. Gene augmentation therapy has proven to be safe and successful in restoring long-term sight in mice, dogs and humans suffering from LCA or RP. Here, we present a comprehensive overview of the genes, mutations and modifier alleles involved in non-syndromic retinal ciliopathies, review the progress in dissecting the associated retinal disease mechanisms and evaluate gene augmentation approaches to antagonize retinal degeneration in these ciliopathies.

PURPOSE. We evaluated the pathogenicity of the G1961E mutation in the ABCA4 gene, and present the range of retinal phenotypes associated with this mutation in homozygosity in a patient cohort with ABCA4-associated phenotypes. METHODS. Patients were enrolled from the ABCA4 disease database at Columbia University or by inquiry from collaborating physicians. Only patients homozygous for the G1961E mutation were enrolled. The entire ABCA4 gene open reading frame, including all exons and flanking intronic sequences, was sequenced in all patients. Phenotype data were obtained from clinical history and examination, fundus photography, infrared imaging, fundus autofluorescence, fluorescein angiography, and spectral domain-optical coherence tomography. Additional functional data were obtained using the full-field electroretinogram, and static or kinetic perimetry. RESULTS. We evaluated 12 patients homozygous for the G1961E mutation. All patients had evidence of retinal pathology consistent with the range of phenotypes observed in ABCA4 disease. The latest age of onset was recorded at 64 years, in a patient diagnosed initially with age-related macular degeneration (AMD). Of 6 patients in whom severe structural (with/ without functional) fundus changes were detected, 5 had additional, heterozygous or homozygous, variants detected in the ABCA4 gene. CONCLUSIONS. Homozygous G1961E mutation in ABCA4 results in a range of retinal pathology. The phenotype usually is at the milder end of the disease spectrum, with severe phenotypes linked to the presence of additional ABCA4 variants. Our report also highlights that milder, late-onset Stargardt disease may be confused with AMD. (Invest Ophthalmol Vis Sci. 2012; 53:4458–4467) DOI:10.1167/iovs.11-9166

Item does not contain fulltext Cone-rod dystrophy (CRD) and retinitis pigmentosa (RP) are clinically and genetically overlapping heterogeneous retinal dystrophies. By using homozygosity mapping in an individual with autosomal-recessive (ar) RP from a consanguineous family, we identified three sizeable homozygous regions, together encompassing 46 Mb. Next-generation sequencing of all exons, flanking intron sequences, microRNAs, and other highly conserved genomic elements in these three regions revealed a homozygous nonsense mutation (c.497T>A [p.Leu166( *)]) in C8orf37, located on chromosome 8q22.1. This mutation was not present in 150 ethnically matched control individuals, single-nucleotide polymorphism databases, or the 1000 Genomes database. Immunohistochemical studies revealed C8orf37 localization at the base of the primary cilium of human retinal pigment epithelium cells and at the base of connecting cilia of mouse photoreceptors. C8orf37 sequence analysis of individuals who had retinal dystrophy and carried conspicuously large homozygous regions encompassing C8orf37 revealed a homozygous splice-site mutation (c.156-2A>G) in two siblings of a consanguineous family and homozygous missense mutations (c.529C>T [p.Arg177Trp]; c.545A>G [p.Gln182Arg]) in siblings of two other consanguineous families. The missense mutations affect highly conserved amino acids, and in silico analyses predicted that both variants are probably pathogenic. Clinical assessment revealed CRD in four individuals and RP with early macular involvement in two individuals. The two CRD siblings with the c.156-2A>G mutation also showed unilateral postaxial polydactyly. These results underline the importance of disrupted ciliary processes in the pathogenesis of retinal dystrophies.

Contains fulltext : 103551.pdf (Publisher’s version ) (Open Access) For many macromolecular NMR ensembles from the Protein Data Bank (PDB) the experiment-based restraint lists are available, while other experimental data, mainly chemical shift values, are often available from the BioMagResBank. The accuracy and precision of the coordinates in these macromolecular NMR ensembles can be improved by recalculation using the available experimental data and present-day software. Such efforts, however, generally fail on half of all NMR ensembles due to the syntactic and semantic heterogeneity of the underlying data and the wide variety of formats used for their deposition. We have combined the remediated restraint information from our NMR Restraints Grid (NRG) database with available chemical shifts from the BioMagResBank and the Common Interface for NMR structure Generation (CING) structure validation reports into the weekly updated NRG-CING database (http://nmr.cmbi.ru.nl/NRG-CING). Eleven programs have been included in the NRG-CING production pipeline to arrive at validation reports that list for each entry the potential inconsistencies between the coordinates and the available experimental NMR data. The longitudinal validation of these data in a publicly available relational database yields a set of indicators that can be used to judge the quality of every macromolecular structure solved with NMR. The remediated NMR experimental data sets and validation reports are freely available online. 01 januari 2012

Contains fulltext : 111029.pdf (Publisher’s version ) (Open Access) PURPOSE: The purpose of this study is to identify the genetic defect in a Turkish family with autosomal recessive retinitis pigmentosa, nanophthalmos, and optic disc drusen. METHODS: Ophthalmological examinations consisted of measuring the best-corrected visual acuity and the refractive error, electroretinography, optical coherence tomography, B-mode ultrasonography, and fundus photography. The involvement of the membrane frizzled-related protein (MFRP) gene in this family was studied with direct DNA sequencing of the coding exons of MFRP and with linkage analysis with microsatellite markers. After MFRP was excluded, genome-wide homozygosity mapping was performed with 250 K single nucleotide polymorphism (SNP) microarrays. Mutation analysis of the crumbs homolog 1 (CRB1) gene was performed with direct sequencing. RESULTS: Ophthalmological evaluation of both affected individuals in the family revealed a decreased axial length (18-19 mm), retinal dystrophy, macular edema, and hyperopia of >+8.0 diopters. Sequencing of MFRP did not reveal any pathogenic changes, and microsatellite marker analysis showed that the chromosomal region did not segregate within the disease in this family. Genome-wide homozygosity mapping using single nucleotide polymorphism microarrays revealed a 28-Mb homozygous region encompassing the CRB1 gene, and direct sequencing disclosed a novel homozygous missense mutation (p.Gly833Asp) in CRB1. CONCLUSIONS: Previous studies associated mutations in the MFRP gene with the syndrome nanophthalmos-retinitis pigmentosa-foveoschisis-optic disc drusen. In this study, we demonstrated that a similar disease complex can be caused by mutations in the CRB1 gene.

Purpose: To determine the genetic defect and to describe the clinical characteristics in patients with retinitis punctata albescens (RPA) and fundus albipunctatus (FAP). Design: Case series/observational study. Participants: We included 13 patients affected by RPA or FAP. Methods: Thirteen patients were collected from 8 families with a retinal dystrophy characterized by tiny, yellow-white dots on funduscopy, typical for FAP or RPA. All patients underwent full ophthalmologic examinations, including visual field assessment. Fundus photography, and electroretinography were performed in 12 patients, and optical coherence tomography and fundus autofluorescence were performed in 4 patients. DNA samples of all patients were screened for mutations in RLBP1 and for mutations in RDH5 in patients who did not carry mutations in RLBP1. DNA samples of 2 sibling pairs of nonconsanguineous families who carried mutations neither in RLBP1 nor in RDH5 were analyzed by genome-wide homozygosity mapping. Sequence analysis was performed of LRAT, a candidate gene in a shared homozygous region. Main Outcome Measures: We assessed DNA sequence variants, best-corrected visual acuity, fundus appearance, visual field measurements, electroretinogram responses, optical coherence tomography, and fundus autofluorescence. Results: A homozygous frameshift mutation was identified in LRAT in 4 patients with RPA. Mutations in RLBP1 were identified in 7 patients with RPA and in 1 patient with FAP and cone dystrophy. One patient had compound heterozygous mutations in RDH5 and suffered from FAP with mild maculopathy. Conclusions: A genetic defect was identified in LRAT as a novel cause of RPA. LRAT is therefore the fourth gene involved in the visual cycle that may cause a white-dot retinopathy. We also revealed that mutations in RLBP1 may lead to FAP with cone dystrophy. Financial Disclosure(s): The authors have no proprietary or commercial interest in any materials discussed in this article. Ophthalmology 2012; 119: 1899-1906 (C) 2012 by the American Academy of Ophthalmology

Item does not contain fulltext The discovery of oncogenes and tumor suppressors as a driver of cancer development has triggered the development of target-specific small molecule anticancer compounds. As exemplified by Imatinib (Gleevec), a specific inhibitor of the Chronic Myeloid Leukemia-associated BCR/ABL kinase, these agents promise impressive activity in clinical trials, with low levels of clinical toxicity. However, such therapy is susceptible to the emergence of drug resistance mainly due to amino acid substitutions in the target protein. Defining the spectrum of such mutations is important for patient monitoring and the design of next-generation inhibitors. Using Imatinib and BCR/ABL as a paradigm for a drug-target pair, we reported a retroviral vector-based screening strategy to identify the spectrum of resistance-conferring mutations, which has helped in designing the next-generation BCR/ABL inhibitors such as Nilotinib, Dasatinib, and Ponatinib. Here we provide a detailed methodology for the screen, which can be generally applied to any drug-target pair.

Item does not contain fulltext Implementation of multiplex ligation-dependent probe amplification (MLPA) for thalassemia causing deletions has lead to the detection of new rearrangements. Knowledge of the exact breakpoint sequences should give more insight into the molecular mechanisms underlying these rearrangements, and would facilitate the design of gap-PCRs. We have designed a custom fine-tiling array with oligonucleotides covering the complete globin gene clusters. We hybridized 27 DNA samples containing newly identified deletions and nine positive controls. We designed specific primers to amplify relatively short fragments containing the breakpoint sequence and analyzed these by direct sequencing. Results from nine positive controls showed that array comparative genomic hybridization (aCGH) is suitable to detect small and large rearrangements. We were able to locate all breakpoints to a region of approximately 2 kb. We designed breakpoint primers for 22 cases and amplification was successful in 19 cases. For 12 of these, the exact locations of the breakpoints were determined. Seven of these deletions have not been reported before. aCGH is a valuable tool for high-resolution breakpoint characterization. The combination of MLPA and aCGH has lead to relatively cheap and easy to perform PCR assays, which might be of use for laboratories as an alternative for MLPA in populations where only a limited number of specific deletions occur with high frequency.

BACKGROUND: Split-hand/foot malformation (SHFM)-also known as ectrodactyly-is a congenital disorder characterised by severe malformations of the distal limbs affecting the central rays of hands and/or feet. A distinct entity termed SHFLD presents with SHFM and long bone deficiency. Mouse models suggest that a defect of the central apical ectodermal ridge leads to the phenotype. Although six different loci/mutations (SHFM1-6) have been associated with SHFM, the underlying cause in a large number of cases is still unresolved. METHODS: High resolution array comparative genomic hybridisation (CGH) was performed in patients with SHFLD to detect copy number changes. Candidate genes were further evaluated for expression and function during limb development by whole mount in situ hybridisation and morpholino knock-down experiments. RESULTS: Array CGH showed microduplications on chromosome 17p13.3, a locus previously associated with SHFLD. Detailed analysis of 17 families revealed that this copy number variation serves as a susceptibility factor for a highly variable phenotype with reduced penetrance, particularly in females. Compared to other known causes for SHFLD 17p duplications appear to be the most frequent cause of SHFLD. A ~11.8 kb minimal critical region was identified encompassing a single gene, BHLHA9, a putative basic loop helix transcription factor. Whole mount in situ hybridisation showed expression restricted to the limb bud mesenchyme underlying the apical ectodermal ridge in mouse and zebrafish embryos. Knock down of bhlha9 in zebrafish resulted in shortening of the pectoral fins. CONCLUSIONS: Genomic duplications encompassing BHLHA9 are associated with SHFLD and non-Mendelian inheritance characterised by a high degree of non-penetrance with sex bias. Knock-down of bhlha9 in zebrafish causes severe reduction defects of the pectoral fin, indicating a role for this gene in limb development.

Contains fulltext : 108895.pdf (Publisher’s version ) (Open Access) PURPOSE: This study investigated the centrosomal protein, 290-KD (CEP290) associated genotype and ocular and extra-ocular phenotype in 18 patients with Leber congenital amaurosis (LCA). METHODS: Eighteen patients with LCA from 14 families with mutations in the CEP290 gene were identified with sequencing or with heteroduplex analysis. Ophthalmic examinations were performed on all patients. Scans of the central nervous system were reassessed in three patients and obtained in two. Renal function was evaluated in all patients. Ultrasonography of the kidneys was performed in six patients. RESULTS: Eight patients (from five families) carried the c.2991+1655A>G mutation homozygously. Nine solitary patients carried this variant combined with a nonsense, frameshift, or splice site mutation on the second allele. One new nonsense mutation was identified: c.1078C>T. Fourteen patients (from 12 families) had been completely blind from birth or had light perception. The best-recorded visual acuity was 20/200. Peripheral fundus changes appeared to be progressive with a relatively preserved posterior pole. Novel ophthalmic features for the CEP290 phenotype were Coats-like exudative vasculopathy in two patients, a small chorioretinal coloboma in one patient, and well defined, small, atrophic spots at the level of the retinal pigment epithelium causing a dot-like appearance in five patients. Some CEP290 patients exhibited systemic abnormalities. We found abnormal proprioception in two patients and mild mental retardation in one. One patient was infertile due to immobile spermatozoa. No renal abnormalities were detected. CONCLUSIONS: CEP290-associated LCA has a severe, progressive, and clinically identifiable phenotype. Distinct extra-ocular findings were noted, which may be attributed to ciliary dysfunction.

Contains fulltext : 98108.pdf (Publisher’s version ) (Open Access) PURPOSE: Retinitis pigmentosa (RP) is a clinically and genetically heterogeneous retinal disorder. Despite tremendous knowledge about the genes involved in RP, little is known about the genetic causes of RP in Indonesia. Here, we aim to identify the molecular genetic causes underlying RP in a small cohort of Indonesian patients, using genome-wide homozygosity mapping. METHODS: DNA samples from affected and healthy individuals from 14 Indonesian families segregating autosomal recessive, X-linked, or isolated RP were collected. Homozygosity mapping was conducted using Illumina 6k or Affymetrix 5.0 single nucleotide polymorphism (SNP) arrays. Known autosomal recessive RP (arRP) genes residing in homozygous regions and X-linked RP genes were sequenced for mutations. RESULTS: In ten out of the 14 families, homozygous regions were identified that contained genes known to be involved in the pathogenesis of RP. Sequence analysis of these genes revealed seven novel homozygous mutations in ATP-binding cassette, sub-family A, member 4 (ABCA4), crumbs homolog 1 (CRB1), eyes shut homolog (Drosophila) (EYS), c-mer proto-oncogene tyrosine kinase (MERTK), nuclear receptor subfamily 2, group E, member 3 (NR2E3) and phosphodiesterase 6A, cGMP-specific, rod, alpha (PDE6A), all segregating in the respective families. No mutations were identified in the X-linked genes retinitis pigmentosa GTPase regulator (RPGR) and retinitis pigmentosa 2 (X-linked recessive; RP2). CONCLUSIONS: Homozygosity mapping is a powerful tool to identify the genetic defects underlying RP in the Indonesian population. Compared to studies involving patients from other populations, the same genes appear to be implicated in the etiology of recessive RP in Indonesia, although all mutations that were discovered are novel and as such may be unique for this population.

Contains fulltext : 108912.pdf (Publisher’s version ) (Open Access) We recently reported a facile method based on the chelation of [(18)F]aluminum fluoride (Al(18)F) by NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid). Here, we present a further optimization of the (18)F labeling of NOTA-octreotide (IMP466). Octreotide was conjugated with the NOTA chelate and was labeled with (18)F in a two-step, one-pot method. The labeling procedure was optimized with regard to the labeling buffer, ionic strength, peptide concentration, and temperature. Radiochemical yield, specific activity, in vitro stability, and receptor affinity were determined. Biodistribution of (18)F-IMP466 was studied in AR42J tumor-bearing mice. In addition, microPET/CT images were acquired. IMP466 was labeled with Al(18)F in a single step with 97% yield in the presence of 80% (v/v) acetonitrile or ethanol. The labeled product was purified by HPLC to remove unlabeled peptide and unbound Al(18)F. The radiolabeling, including purification, was performed for 45 min. Specific activities of 48,000 GBq/mmol could be obtained. (18)F-IMP466 showed a high tumor uptake and excellent tumor-to-blood ratios at 2 h post-injection. In addition, the low bone uptake indicated that the Al(18)F-NOTA complex was stable in vivo. PET/CT scans revealed excellent tumor delineation and specific accumulation in the tumor. Uptake in receptor-negative organs was low. NOTA-octreotide could be labeled with (18)F in quantitative yields using a rapid two-step, one-pot, method. The compound was stable in vivo and showed rapid accretion in SSTR(2)-receptor-expressing AR42J tumors in nude mice. This method can be used to label other NOTA-conjugated compounds such as RGD peptides, GRPR-binding peptides, and Affibody molecules with (18)F. 01 april 2012

Contains fulltext : 97981.pdf (Publisher’s version ) (Closed access) We tested 16 million SNPs, identified through whole-genome sequencing of 457 Icelanders, for association with gout and serum uric acid levels. Genotypes were imputed into 41,675 chip-genotyped Icelanders and their relatives, for effective sample sizes of 968 individuals with gout and 15,506 individuals for whom serum uric acid measurements were available. We identified a low-frequency missense variant (c.1580C>G) in ALDH16A1 associated with gout (OR = 3.12, P = 1.5 x 10(-16), at-risk allele frequency = 0.019) and serum uric acid levels (effect = 0.36 s.d., P = 4.5 x 10(-21)). We confirmed the association with gout by performing Sanger sequencing on 6,017 Icelanders. The association with gout was stronger in males relative to females. We also found a second variant on chromosome 1 associated with gout (OR = 1.92, P = 0.046, at-risk allele frequency = 0.986) and serum uric acid levels (effect = 0.48 s.d., P = 4.5 x 10(-16)). This variant is close to a common variant previously associated with serum uric acid levels. This work illustrates how whole-genome sequencing data allow the detection of associations between low-frequency variants and complex traits.

Two important lines of research have enhanced our understanding of the molecular role of nutrition in influencing behavior. First, exposure to an adverse environment during early life can influence the long-term behavior of the offspring. Second, regulation of the nervous system development and functioning appears to involve epigenetic mechanisms that require a continuous supply of methyl group donors in food. We hypothesized that a maternal diet during pregnancy deficient in methyl donors (MDD) may lead to altered behavior in offspring through permanent changes in hippocampal DNA methylation. We used a rat model of prenatal dietary MDD to test this hypothesis in female offspring as they aged. Prenatal MDD reduced birth weight, litter size, and newborn viability. Aged female offspring of MDD mothers showed increased anxiety and increased learning ability in comparison with control diet group offspring. To explore the role of MDD on epigenetic mechanisms in the brain of adult offspring, we studied expression and methylation of 4 selected genes coding for glucocorticoid receptor, hydroxysteroid dehydrogenase 11 type 2, neuronatin, and reelin proteins in the hippocampus. No major group differences in methylation or expression of the studied genes were detected, except for a significant down-regulation of the reelin gene in the MDD female offspring. The prenatal MDD diet caused intrauterine growth restriction, associated with long-term effects on the behavior of the offspring. However, the observed behavioral differences between the MDD and control diet offspring cannot be explained by epigenetic regulation of the specific genes investigated in this study.

Summary This report presents the conclusions of the X-ray Validation Task Force of the worldwide Protein Data Bank (PDB). The PDB has expanded massively since current criteria for validation of deposited structures were adopted, allowing a much more sophisticated understanding of all the components of macromolecular crystals. The size of the PDB creates new opportunities to validate structures by comparison with the existing database, and the now-mandatory deposition of structure factors creates new opportunities to validate the underlying diffraction data. These developments highlighted the need for a new assessment of validation criteria. The Task Force recommends that a small set of validation data be presented in an easily understood format, relative to both the full PDB and the applicable resolution class, with greater detail available to interested users. Most importantly, we recommend that referees and editors judging the quality of structural experiments have access to a concise summary of well-established quality indicators., Highlights ► Validation criteria used by the PDB for X-ray crystal structures have been reassessed ► Key scores should be presented prominently in an easily understood format ► A concise validation report should be available to referees of papers on crystal structures

Contains fulltext : 96905.pdf (Publisher’s version ) (Closed access) A fundamental challenge in analyzing exome-sequence data is distinguishing pathogenic mutations from background polymorphisms. To address this problem in the context of a genetically heterogeneous disease, retinitis pigmentosa (RP), we devised a candidate-gene prioritization strategy called cis-regulatory mapping that utilizes ChIP-seq data for the photoreceptor transcription factor CRX to rank candidate genes. Exome sequencing combined with this approach identified a homozygous nonsense mutation in male germ cell-associated kinase (MAK) in the single affected member of a consanguineous Turkish family with RP. MAK encodes a cilium-associated mitogen-activated protein kinase whose function is conserved from the ciliated alga, Chlamydomonas reinhardtii, to humans. Mutations in MAK orthologs in mice and other model organisms result in abnormally long cilia and, in mice, rapid photoreceptor degeneration. Subsequent sequence analyses of additional individuals with RP identified five probands with missense mutations in MAK. Two of these mutations alter amino acids that are conserved in all known kinases, and an in vitro kinase assay indicates that these mutations result in a loss of kinase activity. Thus, kinase activity appears to be critical for MAK function in humans. This study highlights a previously underappreciated role for CRX as a direct transcriptional regulator of ciliary genes in photoreceptors. In addition, it demonstrates the effectiveness of CRX-based cis-regulatory mapping in prioritizing candidate genes from exome data and suggests that this strategy should be generally applicable to a range of retinal diseases.

To access publisher full text version of this article. Please click on the hyperlink in Additional Links field. OBJECTIVE: Rare copy number variants have been implicated in different neurodevelopmental disorders, with the same copy number variants often increasing risk of more than one of these phenotypes. In a discovery sample of 22 schizophrenia patients with an early onset of illness (10-15 years of age), the authors observed in one patient a maternally derived 15q11-q13 duplication overlapping the Prader-Willi/Angelman syndrome critical region. This prompted investigation of the role of 15q11-q13 duplications in psychotic illness. METHOD: The authors scanned 7,582 patients with schizophrenia or schizoaffective disorder and 41,370 comparison subjects without known psychiatric illness for copy number variants at 15q11-q13 and determined the parental origin of duplications using methylation-sensitive Southern hybridization analysis. RESULTS: Duplications were found in four case patients and five comparison subjects. All four case patients had maternally derived duplications (0.05%), while only three of the five comparison duplications were maternally derived (0.007%), resulting in a significant excess of maternally derived duplications in case patients (odds ratio=7.3). This excess is compatible with earlier observations that risk for psychosis in people with Prader-Willi syndrome caused by maternal uniparental disomy is much higher than in those caused by deletion of the paternal chromosome. CONCLUSIONS: These findings suggest that the presence of two maternal copies of a fragment of chromosome 15q11.2-q13.1 that overlaps with the Prader-Willi/Angelman syndrome critical region may be a rare risk factor for schizophrenia and other psychoses. Given that maternal duplications of this region are among the most consistent cytogenetic observations in autism, the findings provide further support for a shared genetic etiology between autism and psychosis. Lundbeck Foundation R34-A3243 Danish National Advanced Technology Foundation 001-2009-2 Danish Council for Independent Research in Medical Sciences Danish Psychiatric Research Foundation European Union (EU) LSHM-CT-2006-037761 HEALTH-2007-2.2.1-10-223423 IMI-JU-NewMeds PIAP-GA-2008-218251 National Institutes of Health MH071425 National Genomic Network (NGFNplus) of the German Federal Ministry of Education and Research (BMBF Wellcome Trust, London Medical Research Council, London info:eu-repo/grantAgreement/EC/FP7/218251

Item does not contain fulltext A subset of ciliopathies, including Sensenbrenner, Jeune, and short-rib polydactyly syndromes are characterized by skeletal anomalies accompanied by multiorgan defects such as chronic renal failure and retinitis pigmentosa. Through exome sequencing we identified compound heterozygous mutations in WDR19 in a Norwegian family with Sensenbrenner syndrome. In a Dutch family with the clinically overlapping Jeune syndrome, a homozygous missense mutation in the same gene was found. Both families displayed a nephronophthisis-like nephropathy. Independently, we also identified compound heterozygous WDR19 mutations by exome sequencing in a Moroccan family with isolated nephronophthisis. WDR19 encodes IFT144, a member of the intraflagellar transport (IFT) complex A that drives retrograde ciliary transport. We show that IFT144 is absent from the cilia of fibroblasts from one of the Sensenbrenner patients and that ciliary abundance and morphology is perturbed, demonstrating the ciliary pathogenesis. Our results suggest that isolated nephronophthisis, Jeune, and Sensenbrenner syndromes are clinically overlapping disorders that can result from a similar molecular cause.

Contains fulltext : 97524.pdf (Publisher’s version ) (Open Access) PURPOSE: The present study was designed to determine the association of polymorphisms of the DNA repair genes X-ray cross-complementing group 1 (XRCC1) (c.1316G>A [rs25487]) and xeroderma pigmentosum complementation group D (XPD) (c.2298A>C [rs13181]) with primary open-angle glaucoma (POAG) and primary closed-angle glaucoma (PCAG). METHODS: In this prospective case-control study, polymerase chain reaction-restriction fragment length polymorphism analysis was used to study the association of XRCC1 and XPD with 160 POAG patients, 163 PCAG patients, and 193 unaffected controls. RESULTS: XRCC1 rs25487 was found to be significantly associated specifically with male POAG patients (chi(2) = 13.2 [p = 0.001]), only for the dominant model (odds ratio [OR] = 2.65 [95% confidence interval [CI] = 1.44-4.85], p < 0.005). In addition XPD rs13181 was also found to be associated with male POAG patients (chi(2) = 12.1 [p < 0.005]), for both dominant (OR = 2.44 [95% CI = 1.33-4.47], p < 0.005) as well as recessive model (OR = 3.62 [95% CI = 1.45-9.01], p < 0.01). Combined genotypes of both the genes revealed that the heterozygote AC/GA was significantly associated with the male POAG patients (z = 3.00 [p < 0.001]). The AA/GG genotype was present at a higher frequency in the male controls and the AA/GA in the female controls and could thus have a protective role in males and females, respectively. CONCLUSIONS: We postulate that defects in the DNA repair genes XRCC1 and XPD may possibly be associated with the progression of POAG in male patients of Pakistani origin.

Summary Background Lynch syndrome is caused by germline mutations in MSH2, MLH1, MSH6 , and PMS2 mismatch-repair genes and leads to a high risk of colorectal and endometrial cancer. We previously showed that constitutional 3′ end deletions of EPCAM can cause Lynch syndrome through epigenetic silencing of MSH2 in EPCAM -expressing tissues, resulting in tissue-specific MSH2 deficiency. We aim to establish the risk of cancer associated with such EPCAM deletions. Methods We obtained clinical data for 194 carriers of a 3′ end EPCAM deletion from 41 families known to us at the Radboud University Nijmegen Medical Centre, Nijmegen, Netherlands and compared cancer risk with data from a previously described cohort of 473 carriers from 91 families with mutations in MLH1, MSH2, MSH6 , or a combined EPCAM–MSH2 deletion. Findings 93 of the 194 EPCAM deletion carriers were diagnosed with colorectal cancer; three of the 92 women with EPCAM deletions were diagnosed with endometrial cancer. Carriers of an EPCAM deletion had a 75% (95% CI 65–85) cumulative risk of colorectal cancer before the age of 70 years (mean age at diagnosis 43 years [SD 12]), which did not differ significantly from that of carriers of combined EPCAM–MSH2 deletion (69% [95% CI 47–91], p=0·8609) or mutations in MSH2 (77% [64–90], p=0·5892) or MLH1 (79% [68–90], p=0·5492), but was higher than noted for carriers of MSH6 mutation (50% [38–62], p EPCAM deletions had a 12% [0–27] cumulative risk of endometrial cancer, which was lower than was that noted for carriers of a combined EPCAM–MSH2 deletion (55% [20–90], p MSH2 (51% [33–69], p=0·0006) or MSH6 (34% [20–48], p=0·0309), but did not differ significantly from that noted for MLH1 (33% [15–51], p=0·1193) mutation carriers. This risk seems to be restricted to deletions that extend close to the MSH2 gene promoter. Of 194 carriers of an EPCAM deletion, three had duodenal cancer and four had pancreatic cancer. Interpretation EPCAM deletion carriers have a high risk of colorectal cancer; only those with deletions extending close to the MSH2 promoter have an increased risk of endometrial cancer. These results underscore the effect of mosaic MSH2 deficiency, leading to variable cancer risks, and could form the basis of an optimised protocol for the recognition and targeted prevention of cancer in EPCAM deletion carriers. Funding Sacha Swarttouw-Hijmans Foundation, Dutch Cancer Society, Deutsche Krebshilfe (German Cancer Aid), Hong Kong Cancer Fund, Hungarian Research Grant OTKA, Norwegian EEA Financial Mechanism (Hungarian National Institute of Oncology), and US National Cancer Institute.

Item does not contain fulltext BACKGROUND: The occurrence of the retrovirus xenotropic murine leukemia virus (MLV)-related virus (XMRV) has been reported in prostate tissue of patients with prostate cancer (PrCa). Considering the potential great medical and social relevance of this discovery, we investigated whether this finding could be confirmed in an independent group of Dutch sporadic PrCa cases. METHODS: We investigated the occurrence of XMRV in fresh-frozen PrCa specimens of 74 PrCa patients from The Netherlands. Total RNA and DNA were isolated, subjected to cDNA synthesis, and analyzed by real-time PCR targeting conserved XMRV sequences. RESULTS: Spiking experiments showed that we were able to detect at least 10 copies of XMRV sequences in 100,000 cells by real-time PCR, demonstrating high sensitivity of the assay. XMRV sequences were detected in 3 out of 74 (i.e., 4%) PrCa specimens. The number of XMRV containing cells was extremely low (1 in 600-7,000 cells). This was corroborated by the fact that XMRV could not be detected in consecutive tissue sections of the initial XMRV-positive cases. CONCLUSIONS: XMRV was rarely detected, and at extremely low levels, in sporadic PrCa samples from Dutch patients. When XMRV would play a causal role in prostate carcinogenesis, integration of the provirus could be expected to be present in, at least, a proportion of tumor cells. Therefore, our data do not support the claim that there is an association between XMRV infection and PrCa in Dutch PrCa patients.

Recently the first community-wide assessments of the prediction of the structures of complexes between proteins and small molecule ligands have been reported in the so-called GPCR Dock 2008 and 2010 assessments. In the current review we discuss the different steps along the protein-ligand modeling workflow by critically analyzing the modeling strategies we used to predict the structures of protein-ligand complexes we submitted to the recent GPCR Dock 2010 challenge. These representative test cases, focusing on the pharmaceutically relevant G Protein-Coupled Receptors, are used to demonstrate the strengths and challenges of the different modeling methods. Our analysis indicates that the proper performance of the sequence alignment, introduction of structural adjustments guided by experimental data, and the usage of experimental data to identify protein-ligand interactions are critical steps in the protein-ligand modeling protocol.

Background: Previously, we showed that gene suppression commonly occurs across chromosome 2q14.2 in colorectal cancer, through a process of long-range epigenetic silencing (LRES), involving a combination of DNA methylation and repressive histone modifications. We now investigate whether LRES also occurs in prostate cancer across this 4-Mb region and whether differential DNA methylation of 2q14.2 genes could provide a regional panel of prostate cancer biomarkers. Methods: We used highly sensitive DNA methylation headloop PCR assays that can detect 10 to 25 pg of methylated DNA with a specificity of at least 1:1,000, and chromatin immunoprecipitation assays to investigate regional epigenetic remodeling across 2q14.2 in prostate cancer, in a cohort of 195 primary prostate tumors and 90 matched normal controls. Results: Prostate cancer cells exhibit concordant deacetylation and methylation of histone H3 Lysine 9 (H3K9Ac and H3K9me2, respectively), and localized DNA hypermethylation of EN1, SCTR, and INHBB and corresponding loss of H3K27me3. EN1 and SCTR were frequently methylated (65% and 53%, respectively), whereas INHBB was less frequently methylated. Conclusions: Consistent with LRES in colorectal cancer, we found regional epigenetic remodeling across 2q14.2 in prostate cancer. Concordant methylation of EN1 and SCTR was able to differentiate cancer from normal (P < 0.0001) and improved the diagnostic specificity of GSTP1 methylation for prostate cancer detection by 26%. Impact: For the first time we show that DNA methylation of EN1 and SCTR promoters provide potential novel biomarkers for prostate cancer detection and in combination with GSTP1 methylation can add increased specificity and sensitivity to improve diagnostic potential. Cancer Epidemiol Biomarkers Prev; 20(1); 148–59. ©2011 AACR.

Contains fulltext : 95597.pdf (Publisher’s version ) (Closed access) Through complementary application of SNP genotyping, whole-genome sequencing and imputation in 38,384 Icelanders, we have discovered a previously unidentified sick sinus syndrome susceptibility gene, MYH6, encoding the alpha heavy chain subunit of cardiac myosin. A missense variant in this gene, c.2161C>T, results in the conceptual amino acid substitution p.Arg721Trp, has an allelic frequency of 0.38% in Icelanders and associates with sick sinus syndrome with an odds ratio = 12.53 and P = 1.5 x 10(2). We show that the lifetime risk of being diagnosed with sick sinus syndrome is around 6% for non-carriers of c.2161C>T but is approximately 50% for carriers of the c.2161C>T variant.