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Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) is increasingly used for target discovery in human disease to complement genomic studies. We have assessed the possibilities and limits of 2-D PAGE applied to human colorectal cancer. Up to 10(8) epithelial cells were purified from paired normal and pathological biopsies using Ber-EP4 coated magnetic beads, allowing the elimination of cellular and fluid contaminations. The mean coefficient of variation (CVAR) of repeated 2-D PAGE analysis with silver staining was lying between 20 and 28%. However, only 47% (interrun) to 76% (intrarun) of spots could be matched within a triplicate experiment. Interindividual phenotypic variability was high. Intratumoral phenotypic variability was not found to be significant. When method and tumor variability were added, 90% of CVAR were inferior to 48%. Thus, two-fold up- or down-regulation of protein expression reveals biological significance. Serial paired comparison of 923 proteins in 10 patients showed highly reproducible differences between normal and cancer tissues. Under well defined experimental conditions and after the high variability of the technique has been considered, 2-D PAGE parallel analysis of paired colorectal samples allows patient-specific tumor profiling.

ETV6/TEL is the first transcription factor identified that is specifically required for hematopoiesis within the bone marrow. This gene has been found to have multiple fusion partners of which 16 have been cloned. Fluorescence in situ hybridization (FISH) analysis in a patient with myelodysplastic syndrome (MDS) revealed a t(1;12)(p36;p13) involving ETV6, with the breakpoint in this gene between exon 2 and exon 3. We report here the cloning of a novel ETV6 partner located on 1p36.1, involved in the t(1;12). 3' RACE-PCR from RNA identified a novel sequence fused to exon 2 of ETV6. Database searches localized this sequence in a bacterial artificial chromosome (BAC) mapped to 1p36 by fingerprint analysis. This result was confirmed by FISH using this BAC as probe. 5' and 3' RACE experiments with primers from this novel sequence were carried out on RNA from a healthy donor and identified a novel full-length mRNA, which we named MDS2 (myelodysplastic syndrome 2). RT-PCR experiments were performed on a panel of human cDNAs to analyze the expression pattern of this gene and they revealed four splicing variants. RT-PCR analysis showed that ETV6-MDS2, but not the reciprocal MDS2-ETV6 fusion transcript, was expressed in the bone marrow of the patient. The product of the ETV6-MDS2 fusion transcript predicts a short ETV6 protein containing the first 54 amino acids of ETV6 plus four novel amino acids, lacking both the PTN and the DNA-binding domains. Possible mechanisms to account for the development of MDS in this patient are discussed.

A nomenclature system has recently been suggested for the description of changes (mutations and polymorphisms) in DNA and protein sequences. These nomenclature recommendations have now been largely accepted. However, current rules do not yet cover all types of mutations, nor do they cover more complex mutations. This document lists the existing recommendations and summarizes suggestions for the description of additional, more complex changes. Another version of this paper has been published in Hum Mut 15:7-12, 2000.

The small GTPase Rab4 is implicated in endocytosis in all cell types, but also plays a specific role in some regulated processes. To better understand the role of Rab4 in regulation of vesicular trafficking, we searched for an effector(s) that specifically recognizes its GTP-bound form. We cloned a ubiquitous 69-kDa protein, Rabip4, that behaves as a Rab4 effector in the yeast two-hybrid system and in the mammalian cell. Rabip4 contains two coiled-coil domains and a FYVE-finger domain. When expressed in CHO cells, Rabip4 is present in early endosomes, because it is colocated with endogenous Early Endosome Antigen 1, although it is absent from Rab11-positive recycling endosomes and Rab-7 positive late endosomes. The coexpression of Rabip4 with active Rab4, but not with inactive Rab4, leads to an enlargement of early endosomes. It strongly increases the degree of colocalization of markers of sorting (Rab5) and recycling (Rab11) endosomes with Rab4. Furthermore, the expression of Rabip4 leads to the intracellular retention of a recycling molecule, the glucose transporter Glut 1. We propose that Rabip4, an effector of Rab4, controls early endosomal traffic possibly by activating a backward transport step from recycling to sorting endosomes.

v-E10, a caspase recruitment domain (CARD)-containing gene product of equine herpesvirus 2, is the viral homologue of the bcl-10 protein whose gene was found to be translocated in mucosa-associated lymphoid tissue (MALT) lymphomas. v-E10 efficiently activates the c-jun NH(2)-terminal kinase (JNK), p38 stress kinase, and the nuclear factor (NF)-kappaB transcriptional pathway and interacts with its cellular homologue, bcl-10, via a CARD-mediated interaction. Here we demonstrate that v-E10 contains a COOH-terminal geranylgeranylation consensus site which is responsible for its plasma membrane localization. Expression of v-E10 induces hyperphosphorylation and redistribution of bcl-10 from the cytoplasm to the plasma membrane, a process which is dependent on the intactness of the v-E10 CARD motif. Both membrane localization and a functional CARD motif are important for v-E10-mediated NF-kappaB induction, but not for JNK activation, which instead requires a functional v-E10 binding site for tumor necrosis factor receptor-associated factor (TRAF)6. Moreover, v-E10-induced NF-kappaB activation is inhibited by a dominant negative version of the bcl-10 binding protein TRAF1, suggesting that v-E10-induced membrane recruitment of cellular bcl-10 induces constitutive TRAF-mediated NF-kappaB activation.

The CluSTr (Clusters of SWISS-PROT and TrEMBL proteins) database offers an automatic classification of SWISS-PROT and TrEMBL proteins into groups of related proteins. The clustering is based on analysis of all pairwise comparisons between protein sequences. Analysis has been carried out for different levels of protein similarity, yielding a hierarchical organisation of clusters. The database provides links to InterPro, which integrates information on protein families, domains and functional sites from PROSITE, PRINTS, Pfam and ProDom. Links to the InterPro graphical interface allow users to see at a glance whether proteins from the cluster share particular functional sites. CluSTr also provides cross-references to HSSP and PDB. The database is available for querying and browsing at http://www.ebi.ac.uk/clustr.

To estimate the error rate of the gene expression machinery and its possible age-related increase, we compared the occurrence of polymerase errors during replication and transcription in (A)/(T) runs, in DNA and RNA of young and old individuals and of early- and late-passage cultured fibroblasts. We analyzed three human genes: TPRD, TGFBR2, and ATRX containing stretches of (A)8, (A)10, and (T)13, respectively. The error rate was determined by sequencing 100 cloned PCR or RT-PCR fragments from each DNA and RNA sample. The error rates in replication and transcription increased with the stretch length. The pooled error rates for genomic DNA were: TPRD (A)8, TGFBR2 (A)10, and ATRX (T)13: 1%+/-0.41, 15.8%+/-1.3, and 31.3%+/-2.9, while those for RNA were: 3.8%+/-0.5, 19.3%+/-2.1, and 54.3%+/-1.8, respectively. The deletions of one nucleotide were the most frequent errors. In the replication analysis, a significant difference was found in old versus young individuals for the ATRX (T)13. In the transcription analysis, significantly higher error rates were obtained in old versus young individuals for the TPRD (A)8 and TGFBR2 (A)10. For these genes, the error rate in RNA isolated from fibroblasts was significantly higher than that in blood. The data show a trend of age-related increase in replication/transcription errors; however further studies are necessary to confirm this hypothesis, since the sample size is small. This imperfect fidelity of the gene expression process may explain the evolutionary disadvantage of nucleotide repeats within coding sequences, and that these repeats are targets for mutations in human diseases.

OBJECTIVE: To investigate the effects of culture conditions, serum and specific cytokines such as insulin-like growth factor (IGF) 1 and interleukin (IL) 1alpha on phenotype and cell survival in cultures of Syrian hamster embryonic chondrocyte-like cells (DES4(+).2). METHODS: Proteins and RNA extracted from subconfluent and confluent early- and late-passage DES4(+).2 cells cultured in the presence or absence of serum and IL-1alpha or IGF-1 or both cytokines together were analysed for the expression of chondrocyte-specific genes and for the chondrogenic transcription factor Sox-9 by Western and Northern blotting. Apoptosis was assessed by agarose gel electrophoresis of labelled low-molecular weight DNA extracted from DES4(+).2 cells and another Syrian hamster embryonic chondrocyte-like cell line, 10W(+).1, cultured under the different conditions and treatments. RESULTS: Early passage DES4(+).2 cells expressed chondrocyte-specific molecules such as collagen types alpha1(II) and alpha1(IX), aggrecan, biglycan and link protein and collagen types alpha1(I) and alpha1(X) mRNAs, suggesting a prehypertrophic chondrocyte-like phenotype. The expression of all genes investigated was cell density- and serum-dependent and was low to undetectable in cell populations from later passages. Early-passage DES4(+).2 and 10W(+).1 cells survived when cultured at low cell density, but died by apoptosis when cultured at high cell density in the absence of serum or IGF-1. IGF-1 and IL-1alpha had opposite and antagonistic effects on the chondrocyte phenotype and survival. Whereas IL-1alpha acting alone suppressed cartilage-specific gene expression without significantly affecting cell survival, IGF-1 increased the steady-state mRNA levels and relieved the IL-1alpha-induced suppression of all the chondrocyte-specific genes investigated; it also enhanced chondrocyte survival. Suppression of the chondrocyte phenotype by the inflammatory cytokine IL-1alpha correlated with marked down-regulation of the transcription factor Sox-9, which was relieved by IGF-1. The expression of the Sox9 gene was closely correlated with the expression of the chondrocyte-specific genes under all conditions and treatments. CONCLUSIONS: The results suggest that the effects of cartilage anabolic and catabolic cytokines IGF-1 and IL-1alpha on the expression of the chondrocyte phenotype are mediated by Sox-9. As Sox-9 appears to be essential for matrix production, the potent effect of IL-1alpha in suppressing Sox-9 expression may limit the ability of cartilage to repair during inflammatory joint diseases. Rheumatology (Oxford)

It has been long recognized that highly polymorphic genetic markers can lead to underestimation of divergence between populations when migration is low. Microsatellite loci, which are characterized by extremely high mutation rates, are particularly likely to be affected. Here, we report genetic differentiation estimates in a contact zone between two chromosome races of the common shrew (Sorex araneus), based on 10 autosomal microsatellites, a newly developed Y-chromosome microsatellite, and mitochondrial DNA. These results are compared to previous data on proteins and karyotypes. Estimates of genetic differentiation based on F- and R-statistics are much lower for autosomal microsatellites than for all other genetic markers. We show by simulations that this discrepancy stems mainly from the high mutation rate of microsatellite markers for F-statististics and from deviations from a single-step mutation model for R-statistics. The sex-linked genetic markers show that all gene exchange between races is mediated by females. The absence of male-mediated gene flow most likely results from male hybrid sterility.

Fanconi anemia (FA) is a clinically and genetically heterogeneous disorder. Clinical care is complicated by variable age at onset and severity of hematologic symptoms. Recent advances in the molecular biology of FA have allowed us to investigate the relationship between FA genotype and the nature and severity of the clinical phenotype. Two hundred forty-five patients from all 7 known complementation groups (FA-A to FA-G) were studied. Mutations were detected in one of the cloned FANC genes in 169 patients; in the remainder the complementation group was assigned by cell fusion or Western blotting. A range of qualitative and quantitative clinical parameters was compared for each complementation group and for different classes of mutation. Significant phenotypic differences were found. FA-G patients had more severe cytopenia and a higher incidence of leukemia. Somatic abnormalities were less prevalent in FA-C, but more common in the rare groups FA-D, FA-E, and FA-F. In FA-A, patients homozygous for null mutations had an earlier onset of anemia and a higher incidence of leukemia than those with mutations producing an altered protein. In FA-C, there was a later age of onset of aplastic anemia and fewer somatic abnormalities in patients with the 322delG mutation, but there were more somatic abnormalities in patients with IVS4 + 4A --> T. This study indicates that FA patients with mutations in the FANCG gene and patients homozygous for null mutations in FANCA are high-risk groups with a poor hematologic outcome and should be considered as candidates both for frequent monitoring and early therapeutic intervention. (Blood. 2000;96:4064-4070)

Consistent gene mutation nomenclature is essential for efficient and accurate reporting, testing, and curation of the growing number of disease mutations and useful polymorphisms being discovered in the human genome. While a codified mutation nomenclature system for simple DNA lesions has now been adopted broadly by the medical genetics community, it is inherently difficult to represent complex mutations in a unified manner. In this article, suggestions are presented for reporting just such complex mutations.

The effects of leptin production in ob/ob mice injected with a plasmid expression vector containing mouse leptin cDNA in the tibialis anterior muscle were investigated. A significant reduction in food intake (-18%, p < 0.01) along the experimental period was found after DNA injection, while differences in body weight gain were only significant (-41%, p < 0.05) when determined between days 2.9 of the study. Concerning adipocytes metabolism, there was a significant increase in oxygen consumption in vitro (+34%, p < 0.05) and in basal lipolysis (+151%, p < 0.05) in DNA-injected mice compared to PBS-injected animals. Our results confirm that functional leptin can be produced in muscle and released into the blood stream and give new support to the fact that leptin may have direct auto- or paracrine effects on adipocytes, possible contributing to the weight- and fat-reducing effects of leptin in ob/ob mice.

Fanconi anemia (FA) is an autosomal recessive disorder associated with hypersensitivity to DNA cross-linking agents and bone marrow failure. At least four complementation groups have been defined, and the FA group C gene (FAC) has been cloned. We have screened 76 unrelated FA patients of diverse ethnic and geographic origins and from unknown complementation groups for mutations in the FAC gene either by chemical cleavage mismatch analysis or by single-strand conformational polymorphism (SSCP). Five mutations were detected in four patients (5.3%), including two novel mutations (W22X and L496R). Nine polymorphisms were detected, seven of which have not been described previously (663A G, L190F, IVS6 + 30C T, 1312V, V449M, Q465R, and 1974G A). Six of the nine polymorphisms occurred in patients or controls from the Tswana or Sotho chiefdoms of South Africa and were not found in 50 unrelated European controls. Restriction site assays were established for all 8 pathogenic mutations identified in the FAC gene to date and used to screen a total of 94 unrelated FA patients. This identified only one other group C patient, who was homozygous for the mutation IVS4 + 4A T. This study indicates that the proportion of FA patients from complementation group C is generally likely to be less than 10%. Guidelines for the selection of FA patients for FAC mutation screening are proposed. © 1996 Wiley-Liss, Inc.

SWISS-2DPAGE is a database of proteins identified on two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). The current release contains 343 entries of human, yeast (Saccharomyces cerevisiae) and Escherichia coli origin, as well as virtual entries for each of the protein sequences in the SWISS-PROT database.

Intercellular signaling molecules of the vertebrate hedgehog family and transcription factors of the winged-helix family have been implicated in floor plate development. We have examined the consequences of misexpressing the vertebrate hedgehog gene vhh-1 (sonic hedgehog, shh) and the winged-helix gene HNF-3 beta in the neural plate and neural tube of frog embryos. Misexpression of either of these genes induces floor plate differentiation at ectopic locations. However, ectopic floor plate induction in response to both vhh-1 and HNF-3 beta was temporally and spatially restricted. At neural plate stages, ectopic floor plate differentiation was not detected. After neural tube closure, ectopic floor plate differentiation was detected but was restricted predominantly to the dorsal region of the neural tube. The ability of winged-helix and vertebrate hedgehog genes to induce floor plate differentiation in vivo may, therefore, be constrained by additional signals that specify the time and position of floor plate differentiation.

Normal human adult articular chondrocytes were used to determine how the chondrocyte phenotype is modulated by culture conditions following long-term culture. We report here for the first time that human articular chondrocytes have a lifespan in the range of 34–37 population doublings. While chondrocytes cultured as monolayers displayed a fibroblastoid morphology and grew faster, those cultured as suspensions over agarose adopted a round morphology and formed clusters of cells reminiscent of chondrocyte differentiation in intact cartilage, with little or no DNA synthesis. These morphologies were independent of the age of the culture. Despite, these morphological differences, however, chondrocytes expressed markers at mRNA and protein levels characteristic of cartilage: namely, types II and IX collagens and the large aggregating proteoglycans, aggrecan, versican and link protein, but not syndecan, under both culture conditions. However, they also expressed type I collagen alpha 1(I) and alpha 2(I) chains. It has been suggested that expression of collagen alpha 1(I) by chondrocytes cultured as monolayers is a marker of the loss of the chondrocyte phenotype. However, we show here, using reverse transcriptase/polymerase chain reaction, that normal fresh intact human articular cartilage expresses collagen alpha 1(I). The data show that following long-term culture human articular chondrocytes retain their differentiated characteristics and that cell shape does not correlate with the expression of the chondrocyte phenotype. It is proposed that loss of the chondrocyte phenotype is marked by the loss of one or more cartilage-specific molecules rather than by the appearance of non-cartilage-specific molecules.

Over a period of many years, germ-cell mutagenesis experiments using the mouse specific-locus test have generated numerous radiation- and chemical-induced alleles of the brown (b; Tyrp 1) locus in mouse chromosome 4. We describe here the origin, maintenance and initial molecular characterization of 28 b mutations that are prenatally lethal when homozygous. Each of these mutations is deleted for Tyrp 1 sequences, and each of 25 mutations tested further is deleted for at least one other locus defined by molecular clones previously found to be closely linked to b by interspecific backcross analysis. A panel of DNAs from mice carrying a lethal b mutation and a Mus spretus chromosome 4 was used in the fine structure mapping of these molecularly defined loci. The deletional nature of each of these prenatally lethal mutations is consistent with the hypothesis that the null phenotype at b has an effect only on the quality (color) of eumelanin produced in melanocytes. The resulting deletion map provides a framework on which to build future molecular-genetic and biological analyses of this region of mouse chromosome 4.

During T cell development in the thymus, T cell receptor (TCR) alpha, beta, gamma, and delta genes are rearranged and expressed. TCR rearrangement strictly depends upon the coordinate activity of two recombinase activating genes, Rag-1 and Rag-2. In this study we have followed the expression of these genes at different stages of intrathymic development. The results indicate that there are two periods of high Rag-1 and Rag-2 mRNA expression. The first wave peaks early at the CD25+CD4-CD8-CD3- stage of development and coincides with the initial appearance of transcripts derived from fully rearranged TCR beta, gamma, and delta genes, whereas the second wave occurs later at the CD4+CD8+ stage coincident with full-length TCR alpha mRNA expression. Active downregulation of Rag-1 and Rag-2 mRNA expression appears to occur in vivo between the two peaks of recombinase activity. This phenomenon can be mimicked in vitro in response to artificial stimuli such as phorbol myristate acetate and calcium ionophore. Collectively our data suggest that recombinase expression is actively regulated during early thymus development independently of cell surface expression of a mature heterodimeric TCR protein complex.

SWISS-PROT is an annotated protein sequence database established in 1986 and maintained collaboratively, since 1988, by the Department of Medical Biochemistry of the University of Geneva and the EMBL Data Library

We have cloned and sequenced mouse cDNAs corresponding to a third member of a family of melanocyte-specific mRNAs, which encode tyrosinase and related proteins. This new member, tyrosinase-related protein-2 (TRP-2), has approximately 40% amino acid identity with the two other proteins in the family and has the same structural features including two copper binding sites, two cysteine-rich regions, a signal peptide and a transmembrane domain. We now show that one of the cysteine-rich regions in this protein family is an 'EGF-like' repeat found in many extracellular and cell surface proteins. The gene encoding TRP-2 maps to mouse chromosome 14, in the region of the coat colour mutation slaty. We show that the TRP-2 of slaty mice has a single amino acid difference from wild-type TRP-2; a substitution of glutamine for arginine in the first copper binding site. TRP-2 is the much sought melanogenic enzyme DOPAchrome tautomerase (DT), which catalyses the conversion of DOPAchrome to 5,6,dihydroxyindole-2-carboxylic acid. Extracts from mice homozygous for the slaty mutation have a 3-fold or more reduction in DT activity, indicating that TRP-2/DT is encoded at the slaty locus, and the missense mutation reduces but does not abolish the enzyme activity.

The chloroplast gene psaC encoding the iron sulfur protein of photosystem I (PSI) from the green alga Chlamydomonas reinhardtii has been cloned and characterized. The deduced amino acid sequence is highly related to that of higher plants and cyanobacteria. Using a particle gun, wild type C. reinhardtii cells have been transformed with a plasmid carrying the psaC gene disrupted by an aadA gene cassette designed to express spectinomycin/streptomycin resistance in the chloroplast. Transformants selected on plates containing acetate as a reduced carbon source and spectinomycin are unable to grow on minimal medium lacking acetate and are deficient in PSI activity. Southern blot analysis of total cell DNA of the transformants shows that the wild type psaC gene has been replaced by the interrupted psaC gene through homologous recombination. While authentic transcripts of the psaC gene are no longer detected, aadA gives rise to a few transcripts in the transformants. Biochemical analysis indicates that neither PSI reaction center subunits nor the seven small subunits belonging to PSI accumulate stably in the thylakoid membranes of the transformants. Pulse-chase labeling of cell proteins shows that the PSI reaction center subunits are synthesized normally but turn over rapidly in the transformants. We conclude that the iron sulfur binding protein encoded by the psaC gene is an essential component, both for photochemical activity and for stable assembly of PSI. The present study suggests that any chloroplast gene encoding a component of the photosynthetic apparatus can be disrupted in C. reinhardtii using the strategy described.

We have determined the exon structure of the mouse tyrosinase-related protein-1 (TRP-1) gene. The gene is only 15kb in length, but contains seven introns, in contrast to the tyrosinase gene which is almost 100kb long with only four introns. Only two introns are located in homologous positions in both genes. Intron I of TRP-1 has three alternative 5' splice sites clustered within 21bp, which all splice to the same 3' site. Intron V has a very unusual 5' splice site, which has the dinucleotide GC rather than the conventional GT. We show that as little as 370bp of 5'-flanking DNA is sufficient to direct cell-specific expression of the chloramphenicol acetyl transferase gene. The flanking DNA of TRP-1, unlike tyrosinase, does not contain a TATA box or a CCAAT box. Both mouse genes, however, share an 11bp sequence, also found in human tyrosinase, which we suggest may be a melanocyte-specific promoter element.

The mouse brown locus encodes a putative membrane-bound metalloenzyme, tyrosinase-related protein-1 (TRP-1). We have examined the effect on mRNA expression of the locus of a number of mutant alleles. The common null mutant allele, brown, produces wild-type levels of TRP-1 mRNA, which is nonfunctional. Another recessive allele, cordovan-Harwell, has an intermediate, dark-brown phenotype and produces only very low levels of presumably normal TRP-1 mRNA. Two dominant alleles appear to act by killing the melanocyte in which they are expressed. One of them, Light, has normal size and amounts of TRP-1 mRNA. The other, White-based brown, produces no detectable TRP-1 mRNA. It has a gross DNA rearrangement at the 5' end, and we speculate that this results in activation of transcription of sequences not usually seen in melanocytes, and that this is toxic to the cell. The relationship between phenotype and molecular structure at the locus is discussed, and we draw some general principles applicable to other developmental genes.

The murine b locus encodes the tyrosinase related protein, TRP-1, a putative membrane-bound, copper-containing enzyme having about 40% amino acid identity with tyrosinase. The protein is essential for production of black rather than brown hair pigment. We show that skin of mutant brown mice contains the same amount of TRP-1 mRNA as wild type. On sequencing the coding region of the mutant mRNA we find four nucleotide differences from the wild-type (Black) sequence. Two of these differences result in different amino acid residues encoded by the brown allele. By sequencing the TRP-1 gene from a mouse in which a reversion from brown to Black has been induced by ethylnitrosourea we are able to show that only one of these amino acid changes, which substitutes a tyrosine for a conserved cysteine, is the cause of the brown phenotype. This mutation is adjacent to another cysteine at which, in the analogous position in tyrosinase a mutation results in the albino phenotype. The sequence of the revertant is the first report of DNA sequence of an ethylnitrosourea-induced genetic change in mouse.

Contains fulltext : 136472.pdf (Publisher’s version ) (Closed access) Age at menarche is a marker of timing of puberty in females. It varies widely between individuals, is a heritable trait and is associated with risks for obesity, type 2 diabetes, cardiovascular disease, breast cancer and all-cause mortality. Studies of rare human disorders of puberty and animal models point to a complex hypothalamic-pituitary-hormonal regulation, but the mechanisms that determine pubertal timing and underlie its links to disease risk remain unclear. Here, using genome-wide and custom-genotyping arrays in up to 182,416 women of European descent from 57 studies, we found robust evidence (P < 5 x 10(-8)) for 123 signals at 106 genomic loci associated with age at menarche. Many loci were associated with other pubertal traits in both sexes, and there was substantial overlap with genes implicated in body mass index and various diseases, including rare disorders of puberty. Menarche signals were enriched in imprinted regions, with three loci (DLK1-WDR25, MKRN3-MAGEL2 and KCNK9) demonstrating parent-of-origin-specific associations concordant with known parental expression patterns. Pathway analyses implicated nuclear hormone receptors, particularly retinoic acid and gamma-aminobutyric acid-B2 receptor signalling, among novel mechanisms that regulate pubertal timing in humans. Our findings suggest a genetic architecture involving at least hundreds of common variants in the coordinated timing of the pubertal transition.

Body fat distribution is a risk factor for adverse cardiovascular health consequences. We analyzed the association of body fat distribution, assessed by waist-to-hip ratio adjusted for body mass index, with 228,985 predicted coding and splice site variants available on exome arrays in up to 344,369 individuals from five major ancestries (discovery) and 132,177 European-ancestry individuals (validation). We identified 15 common (minor allele frequency, MAF ≥ 5%) and 9 low frequency or rare (MAF < 5%) coding novel variants. Pathway/gene set enrichment analyses identified lipid particle, adiponectin, abnormal white adipose tissue physiology, and bone development and morphology as important contributors to fat distribution, while cross-trait associations highlight cardiometabolic traits. In functional follow-up analyses, specifically in Drosophila RNAi-knockdowns, we observed a significant increase in the total body triglyceride levels for two genes (DNAH10 and PLXND1). We implicate novel genes in fat distribution, stressing the importance of interrogating low-frequency and protein-coding variants., Editorial summary: A trans-ethnic exome-wide association study for body fat distribution identifies protein-coding variants that are significantly associated with waist-to-hip ratio adjusted for body mass index.

SWISS-PROT is a curated protein sequence database which strives to provide a high level of annotation, a minimal level of redundancy and high level of integration with other databases. Ongoing genome sequencing projects have dramatically increased the number of protein sequences to be incorporated into SWISS-PROT. Since we do not want to dilute the quality standards of SWISS-PROT by incorporating sequences without proper sequence analysis and annotation, we cannot speed up the incorporation of new incoming data indefinitely. However, as we also want to make the sequences available as fast as possible, we introduced TREMBL (TRanslation of EMBL nucleotide sequence database), a supplement to SWISS-PROT. TREMBL consists of computer-annotated entries in SWISS-PROT format derived from the translation of all coding sequences (CDS) in the EMBL nucleotide sequence database, except for CDS already included in SWISS-PROT. While TREMBL is already of immense value, its computer-generated annotation does not match the quality of SWISS-PROTs. The main difference is in the protein functional information attached to sequences. With this in mind, we are dedicating substantial effort to develop and apply computer methods to enhance the functional information attached to TREMBL entries.

Background: The GENCODE consortium was formed to identify and map all protein-coding genes within the ENCODE regions. This was achieved by a combination of initial manual/nannotation by the HAVANA team, experimental validation by the GENCODE consortium and a refinement of the annotation based on these experimental results./nResults: The GENCODE gene features are divided into eight different categories of which only/nthe first two (known and novel coding sequence) are confidently predicted to be protein-coding/ngenes. 5’ rapid amplification of cDNA ends (RACE) and RT-PCR were used to experimentally/nverify the initial annotation. Of the 420 coding loci tested, 229 RACE products have been/nsequenced. They supported 5’ extensions of 30 loci and new splice variants in 50 loci. In addition,/n46 loci without evidence for a coding sequence were validated, consisting of 31 novel and 15/nputative transcripts. We assessed the comprehensiveness of the GENCODE annotation by/nattempting to validate all the predicted exon boundaries outside the GENCODE annotation. Out/nof 1,215 tested in a subset of the ENCODE regions, 14 novel exon pairs were validated, only two/nof them in intergenic regions./nConclusions: In total, 487 loci, of which 434 are coding, have been annotated as part of the/nGENCODE reference set available from the UCSC browser. Comparison of GENCODE/nannotation with RefSeq and ENSEMBL show only 40% of GENCODE exons are contained within/nthe two sets, which is a reflection of the high number of alternative splice forms with unique/nexons annotated. Over 50% of coding loci have been experimentally verified by 5’ RACE for/nEGASP and the GENCODE collaboration is continuing to refine its annotation of 1% human/ngenome with the aid of experimental validation.

The correct subcellular distribution of proteins establishes the complex morphology and function of neurons. Fluorescence microscopy techniques are invaluable to investigate subcellular protein distribution, but they suffer from the limited ability to efficiently and reliably label endogenous proteins with fluorescent probes. We developed ORANGE: Open Resource for the Application of Neuronal Genome Editing, which mediates targeted genomic integration of epitope tags in rodent dissociated neuronal culture, in organotypic slices, and in vivo. ORANGE includes a knock-in library for in-depth investigation of endogenous protein distribution, viral vectors, and a detailed two-step cloning protocol to develop knock-ins for novel targets. Using ORANGE with (live-cell) superresolution microscopy, we revealed the dynamic nanoscale organization of endogenous neurotransmitter receptors and synaptic scaffolding proteins, as well as previously uncharacterized proteins. Finally, we developed a mechanism to create multiple knock-ins in neurons, mediating multiplex imaging of endogenous proteins. Thus, ORANGE enables quantification of expression, distribution, and dynamics for virtually any protein in neurons at nanoscale resolution., This study describes the development of a genome editing toolbox (ORANGE) for endogenous tagging of proteins in neurons. This open resource allows the investigation of protein localization and dynamics in neurons using live-cell and super-resolution imaging techniques.