ALBERT

Description: Transformation of refractory cratonic mantle into more fertile lithologies is the key to the fate of cratonic lithosphere. This process has been extensively studied in the eastern North China Craton (NCC) while that of its western part is still poorly constrained. A comprehensive study of newly-found pyroxenite xenoliths from the Langshan area, in the northwestern part of this craton is integrated with a regional synthesis of pyroxenite and peridotite xenoliths to constrain the petrogenesis of the pyroxenites and provide an overview of the processes involved in the modification of the deep lithosphere. The Langshan pyroxenites are of two types, high-Mg# [Mg2+/(Mg2++Fe2+)*100 = ∼ 90, atomic ratios] olivine-bearing websterites with high equilibration temperatures (880 ∼ 970 oC), and low-Mg# (70 ∼ 80) plagioclase-bearing websterites with low equilibration temperatures (550 ∼ 835 oC). The high-Mg# pyroxenites show trade-off abundances of olivine and orthopyroxene, highly depleted bulk Sr-Nd (ƐNd = +11.41, 87Sr/86Sr = ∼0.7034) and low clinopyroxene Sr isotopic ratios (mean 87Sr/86Sr = ∼0.703). They are considered to reflect the reaction of mantle peridotites with silica-rich silicate melts derived from the convective mantle. Their depletion in fusible components (e.g., FeO, TiO2 and Na2O) and progressive exhaustion of incompatible elements suggest melt extraction after their formation. The low-Mg# pyroxenites display layered structures, convex-upward rare earth element patterns, moderately enriched bulk Sr-Nd isotopic ratios (ƐNd = -14.20 ∼ -16.74, 87Sr/86Sr = 0.7070 ∼ 0.7078) and variable clinopyroxene Sr-isotope ratios (87Sr/86Sr = 0.706-0.711). They are interpreted to be crustal cumulates from hypersthene-normative melts generated by interaction between the asthenosphere and heterogeneous lithospheric mantle. Combined with studies on regional peridotite xenoliths, it is shown that the thinning and refertilization of the lithospheric mantle was accompanied by crustal rejuvenation and that such processes occurred ubiquitously in the northwestern part of the NCC. A geodynamic model is proposed for the evolution of the deep lithosphere, which includes long-term mass transfer through a mantle wedge into the deep crust from the Paleozoic to the Cenozoic, triggered by subduction of the Paleo-Asian ocean and the Late Mesozoic lithospheric extension of eastern Asia.

Description: The orogenic development after the continental collision between Laurussia and Gondwana, led to two contrasting associations of mantle-derived magmatic rocks on the territory of the Bohemian Massif: (i) a 340–310 Ma lamprophyre-lamproite orogenic association and (ii) a 300–275 Ma lamprophyre association of anorogenic affinity. Major types of potassic mantle-derived magmatic rocks recognised in the orogenic and anorogenic associations include: (i) calc-alkaline to alkaline lamprophyres, (ii) alkaline “orthopyroxene minettes” (and geochemically related rocks), and (iii) peralkaline lamproites. These three types significantly differ with respect to mineral, whole-rock and Sr–Nd–Pb–Li isotope composition, and spatial distribution. The calc-alkaline lamprophyres occur throughout the entire Saxo-Thuringian and Moldanubian zones, whereas the different types of malte-derived potassic rocks are spatially restricted to particular zones. Rocks of the Carboniferous lamprophyre-lamproite orogenic association are characterised by variable negative εNd(i) and variably radiogenic Sr(i), whereas the rocks of the Permian lamprophyre association of anorogenic affinity are characterised by positive εNd(i) and relatively young depleted-mantle Nd-model ages reflecting increasing input from upwelling asthenospheric mantle. The small variation in the Pb isotopic composition of post-collisional potassic mantle-derived magmatic rocks (of both the orogenic and anorogenic series) implies that the Pb budget of the mantle beneath the Bohemian Massif is dominated by the same crust-derived material, which itself may include material derived from several sources. The source rocks of “orthopyroxene minettes” are characterised by isotopically light (“eclogitic”) Li and strongly radiogenic (crustal) Sr and may have been metasomatised by high-pressure fluids along the edge of a subduction zone. In contrast, the strongly Al2O3 and CaO depleted mantle source of the lamproites is characterised by isotopically heavy Li and high SiO2 and extreme K2O contents. This mantle source may have been metasomatised predominantly by melts. The mantle source of the lamprophyres may have undergone metasomatism by both fluids and melts.

Description: The Lu–Hf isotope system and Sr–Nd–Hf–Os isotope systematics of mantle rocks are capable of unravelling the early processes in collision belts, especially in a hot subduction context where the Sm–Nd and U–Pb systems in crustal rocks are prone to resetting owing to high temperatures and interaction with melts during exhumation. To improve models of the Devonian–Carboniferous evolution of the Bohemian Massif, we investigated in detail mafic and ultramafic rocks (eclogite, pyroxenite, and peridotite) from the ultrahigh-pressure and ultrahigh-temperature Kutná Hora Crystalline Complex (KHCC: Úhrov, Bečváry, Doubrava, and Spačice localities). Petrography, multiphase solid inclusions, major and trace element compositions of rocks and minerals, and radiogenic isotopic data document contrasting sources and protoliths as well as effects of subduction-related processes for these rocks. The Úhrov peridotite has a depleted composition corresponding to the suboceanic asthenospheric mantle, whereas Bečváry and Doubrava peridotites represent lithospheric mantle that underwent melt refertilization by basaltic and SiO2-undersaturated melts, respectively. Multiphase solid inclusions enclosed in garnet from Úhrov and Bečváry peridotites represent trapped H2O ± CO2-bearing metasomatizing agents and Fe–Ti-rich melts. The KHCC eclogites either formed by high-pressure crystal accumulation from mantle-derived basaltic melts (Úhrov) or represent a fragment of mid-ocean ridge basalt-like gabbroic cumulate (Spačice) and crustal-derived material (Doubrava) both metamorphosed at high P–T conditions. The Lu–Hf age of 395 ± 23 Ma obtained for the Úhrov peridotite reflects garnet growth related to burial of the asthenospheric mantle during subduction of the oceanic slab. By contrast, Spačice and Doubrava eclogites yield younger Lu–Hf ages of ∼350 and 330 Ma, respectively, representing mixed ages as demonstrated by the strong granulite-facies overprint and trace element zoning in garnet grains. We propose a refined model for the Early Variscan evolution of the Bohemian Massif starting with the subduction of the oceanic crust (Saxothuringian ocean) and associated oceanic asthenospheric mantle (Úhrov) beneath the Teplá–Barrandian at ≥380 Ma, which was responsible for melt refertilization of the associated mantle wedge (Bečváry, Doubrava). This was followed by continental subduction (∼370–360 Ma?) accompanied by the oceanic slab break-off and incorporation of the upwelling asthenospheric mantle into the Moldanubian lithospheric mantle and subsequent coeval exhumation of mantle and crustal rocks at ∼350–330 Ma.

Description: The Changning–Menglian orogenic belt (CMOB) in the southeastern Tibetan Plateau is an important link between the Longmu Co–Shuanghu suture (LCSS) in the northern Tibetan Plateau and the Chiang Mai–Inthanon and Bentong–Raub sutures in Thailand and Peninsular Malaysia. These belts and sutures are generally regarded as containing the remnants of the oceanic crust of the Palaeo-Tethys that formed by seafloor spreading as a result of the separation of Gondwana- and Eurasia-derived blocks during the Middle Cambrian. In this paper we report the first discovery of abundant unaltered and retrograde eclogites that occur as irregular lenses and blocks in metasedimentary rocks of the CMOB, and these eclogites form an elongate and almost north–south-trending high-pressure (HP)–ultrahigh-pressure (UHP) metamorphic belt that is ∼200 km long and ∼50 km wide. The newly discovered phengite/talc/epidote–glaucophane eclogites, lawsonite–talc–phengite eclogites, dolomite/magnesite–kyanite eclogites and phengite–kyanite-bearing retrograde eclogites have enriched (E-) and normal mid-ocean ridge basalt (N-MORB)-like affinities and mainly positive as well as some negative whole-rock εNd values (–4·34 to +7·89), which suggest an enriched and depleted oceanic lithosphere source for their protoliths. Magmatic zircons separated from the epidote–glaucophane, magnesite–kyanite and (phengite–kyanite-bearing) retrograde eclogites gave protolith ages of 317–250 Ma, which fit well within the time frame of the opening of the Palaeo-Tethys during the Middle Cambrian and its closure during the Triassic. Abundant metamorphic zircons in the eclogites indicate a Triassic metamorphic event related to the subduction of the Palaeo-Tethys oceanic crust from 235 to 227 Ma. Taking into account previous isotopic age data, we now establish the periods of Early–Middle Triassic (246–227 Ma) and Late Triassic (222–209 Ma) as the ages of subduction and exhumation of the Palaeo-Tethyan oceanic crust, respectively. Thermodynamic modelling revealed that the eclogites record distinct HP–UHP peak metamorphic conditions of 23·0–25·5 kbar and 582–610 °C for the phengite–glaucophane eclogites, 24·0–25·5 kbar and 570–586 °C for the talc–glaucophane eclogites, 29·0–31·0 kbar and 675–712 °C for the dolomite–kyanite eclogites, and 30·0–32·0 kbar and 717–754 °C for the magnesite–kyanite eclogites. These P–T estimates and geochronological data indicate that the Palaeo-Tethys oceanic slab was subducted to different mantle depths from 75 km down to 95 km, forming distinct types of eclogite with a variety of peak eclogite-facies mineral assemblages. The eclogites consistently record clockwise metamorphic P–T–t paths characterized by a heating–compression prograde loop under a low geothermal gradient of 5–10 °C km–1, indicating the rapid subduction of cold oceanic crust at a rate of 4·5–6·0 km Ma–1, followed by isothermal or cooling–decompressive retrogression and exhumation at an average rate of 3·2–4·2 km Ma–1. The newly discovered eclogites of the CMOB with their signatures of ocean-crust subduction are petrologically, geochemically and geochronologically comparable with those of the LCSS, providing powerful support for the idea that a nearly 2000 km long HP–UHP eclogite belt extends from the northern Tibetan Plateau to the southeastern Tibetan Plateau, and that it represents the main boundary suture of the Palaeo-Tethyan domain. These results have far-reaching implications for the tectonic framework and complex metamorphic evolution of the Palaeo-Tethyan domain.

Description: Replacive symplectites (vermicular intergrowths of two or more minerals) are an important feature of layered igneous intrusions, recording evidence of late-stage reactions between interstitial liquid and crystals. They are common throughout the Layered Series of the 564 Ma Sept Iles layered intrusion in Quebec, Canada, and fall into three types: oxy-symplectites, ‘Type I’ symplectites, and ‘Type II’ symplectites. Oxy-symplectites are comprised of magnetite and orthopyroxene, nucleate on olivine primocrysts, and form via the reaction Olivine + O2 → Orthopyroxene + Magnetite; Type I symplectites (of which there are 3 distinct categories) are comprised of anorthitic plagioclase with pyroxene, amphibole, or olivine vermicules, grow from primocryst oxide grains, and replace primary plagioclase; and Type II symplectites (of which there are 2 distinct categories) are comprised of anorthitic plagioclase with orthopyroxene ± amphibole vermicules, grow from primocryst olivine grains, and replace primocryst plagioclase. Rare symplectites composed of biotite and plagioclase are also present. Symplectite growth occurred at 700-1030 °C with pressure constraints of 1-2 kbar. We propose that Type I symplectites, and some Type II symplectites, formed from interaction of primocrysts with residual Fe-rich liquid as a consequence of differential loss of an immiscible Si-rich liquid conjugate from the crystal mush. However, redistribution and concentration of hydrous fluids in incompletely solidified rock, or an increase in water activity of the interstitial melt, may be more plausible processes responsible for the formation of replacive symplectites comprising abundant hydrous mineral assemblages.

Description: Huntington's disease (HD) is a hereditary neurodegenerative disorder characterized by brain atrophy particularly in striatum leading to personality changes, chorea and dementia. Glycogen synthase kinase-3 (GSK-3) is a serine/threonine kinase in the crossroad of many signaling pathways that is highly pleiotropic as it phosphorylates more than hundred substrates including structural, metabolic, and signaling proteins. Increased GSK-3 activity is believed to contribute to the pathogenesis of neurodegenerative diseases like Alzheimer's disease and GSK-3 inhibitors have been postulated as therapeutic agents for neurodegeneration. Regarding HD, GSK-3 inhibitors have shown beneficial effects in cell and invertebrate animal models but no evident efficacy in mouse models. Intriguingly, those studies were performed without interrogating GSK-3 level and activity in HD brain. Here we aim to explore the level and also the enzymatic activity of GSK-3 in the striatum and other less affected brain regions of HD patients and of the R6/1 mouse model to then elucidate the possible contribution of its alteration to HD pathogenesis by genetic manipulation in mice. We report a dramatic decrease in GSK-3 levels and activity in striatum and cortex of HD patients with similar results in the mouse model. Correction of the GSK-3 deficit in HD mice, by combining with transgenic mice with conditional GSK-3 expression, resulted in amelioration of their brain atrophy and behavioral motor and learning deficits. Thus, our results demonstrate that decreased brain GSK-3 contributes to HD neurological phenotype and open new therapeutic opportunities based on increasing GSK-3 activity or attenuating the harmful consequences of its decrease.

Description: Spinocerebellar ataxia type 6 (SCA6) is dominantly inherited neurodegenerative disease, caused by an expansion of CAG repeat encoding a polyglutamine (PolyQ) tract in the Ca v 2.1 voltage-gated calcium channel. Its key pathological features include selective degeneration of the cerebellar Purkinje cells (PCs), a common target for PolyQ-induced toxicity in various SCAs. Mutant Ca v 2.1 confers toxicity primarily through a toxic gain-of-function mechanism; however, its molecular basis remains elusive. Here, we studied the cerebellar gene expression patterns of young Sca6 -MPI 118Q/118Q knockin (KI) mice, which expressed mutant Ca v 2.1 from an endogenous locus and recapitulated many phenotypic features of human SCA6. Transcriptional signatures in the MPI 118Q/118Q mice were distinct from those in the Sca1 154Q/2Q mice, a faithful SCA1 KI mouse model. Temporal expression profiles of the candidate genes revealed that the up-regulation of genes associated with microglial activation was initiated before PC degeneration and was augmented as the disease progressed. Histological analysis of the MPI 118Q/118Q cerebellum showed the predominance of M1-like pro-inflammatory microglia and it was concomitant with elevated expression levels of tumor necrosis factor, interleukin-6, Toll-like receptor (TLR) 2 and 7. Genetic ablation of MyD88, a major adaptor protein conveying TLR signaling, altered expression patterns of M1/M2 microglial phenotypic markers in the MPI 118Q/118Q cerebellum. More importantly, it ameliorated PC loss and partially rescued motor impairments in the early disease phase. These results suggest that early neuroinflammatory response may play an important role in the pathogenesis of SCA6 and its modulation could pave the way for slowing the disease progression during the early stage of the disease.

Description: Adaptor proteins (AP 1–5) are heterotetrameric complexes that facilitate specialized cargo sorting in vesicular-mediated trafficking. Mutations in AP5Z1 , encoding a subunit of the AP-5 complex, have been reported to cause hereditary spastic paraplegia (HSP), although their impact at the cellular level has not been assessed. Here we characterize three independent fibroblast lines derived from skin biopsies of patients harbouring nonsense mutations in AP5Z1 and presenting with spastic paraplegia accompanied by neuropathy, parkinsonism and/or cognitive impairment. In all three patient-derived lines, we show that there is complete loss of AP-5 protein and a reduction in the associated AP-5 µ5 protein. Using ultrastructural analysis, we show that these patient-derived lines consistently exhibit abundant multilamellar structures that are positive for markers of endolysosomes and are filled with aberrant storage material organized as exaggerated multilamellar whorls, striated belts and ‘fingerprint bodies’. This phenotype can be replicated in a HeLa cell culture model by siRNA knockdown of AP-5 . The cellular phenotype bears striking resemblance to features described in a number of lysosomal storage diseases (LSDs). Collectively, these findings reveal an emerging picture of the role of AP-5 in endosomal and lysosomal homeostasis, illuminates a potential pathomechanism that is relevant to the role of AP-5 in neurons and expands the understanding of recessive HSPs. Moreover, the resulting accumulation of storage material in endolysosomes leads us to propose that AP-5 deficiency represents a new type of LSDs.

Description: Cleft palate is a common birth defect in humans. Therefore, understanding the molecular genetics of palate development is important from both scientific and medical perspectives. Lhx6 and Lhx8 encode LIM homeodomain transcription factors, and inactivation of both genes in mice resulted in profound craniofacial defects including cleft secondary palate. The initial outgrowth of the palate was severely impaired in the mutant embryos, due to decreased cell proliferation. Through genome-wide transcriptional profiling, we discovered that p57 Kip2 ( Cdkn1c ), encoding a cell cycle inhibitor, was up-regulated in the prospective palate of Lhx6 –/– ;Lhx8 –/– mutants. p57 Kip2 has been linked to Beckwith–Wiedemann syndrome and IMAGe syndrome in humans, which are developmental disorders with increased incidents of palate defects among the patients. To determine the molecular mechanism underlying the regulation of p57 Kip2 by the Lhx genes, we combined chromatin immunoprecipitation, in silico search for transcription factor-binding motifs, and in vitro reporter assays with putative cis-regulatory elements. The results of these experiments indicated that LHX6 and LHX8 regulated p57 Kip2 via both direct and indirect mechanisms, with the latter mediated by Forkhead box (FOX) family transcription factors. Together, our findings uncovered a novel connection between the initiation of palate development and a cell cycle inhibitor via LHX. We propose a model in which Lhx6 and Lhx8 negatively regulate p57 Kip2 expression in the prospective palate area to allow adequate levels of cell proliferation and thereby promote normal palate development. This is the first report elucidating a molecular genetic pathway downstream of Lhx in palate development.

Description: Keratoconus is a degenerative eye condition which results from thinning of the cornea and causes vision distortion. Treatments such as ultraviolet (UV) cross-linking have proved effective for management of keratoconus when performed in early stages of the disease. The central corneal thickness (CCT) is a highly heritable endophenotype of keratoconus, and it is estimated that up to 95% of its phenotypic variance is due to genetics. Genome-wide association efforts of CCT have identified common variants (i.e. minor allele frequency (MAF) 〉5%). However, these studies typically ignore the large set of exonic variants whose MAF is usually low. In this study, we performed a CCT exome-wide association analysis in a sample of 1029 individuals from a population-based study in Western Australia. We identified a genome-wide significant exonic variant rs121908120 ( P = 6.63 x 10 –10 ) in WNT10A . This gene is 437 kb from a gene previously associated with CCT ( USP37 ). We showed in a conditional analysis that the WNT10A variant completely accounts for the signal previously seen at USP37 . We replicated our finding in independent samples from the Brisbane Adolescent Twin Study, Twin Eye Study in Tasmania and the Rotterdam Study. Further, we genotyped rs121908120 in 621 keratoconus cases and compared the frequency to a sample of 1680 unscreened controls from the Queensland Twin Registry. We found that rs121908120 increases the risk of keratoconus two times (odds ratio 2.03, P = 5.41 x 10 –5 ).

Description: Somatic cell cytokinesis was shown to involve the insertion of sphingolipids (SLs) to midbodies prior to abscission. Spermatogenic midbodies transform into stable intercellular bridges (ICBs) connecting clonal daughter cells in a syncytium. This process requires specialized SL structures. (1) Using high resolution-mass spectrometric imaging, we show in situ a biphasic pattern of SL synthesis with testis-specific anchors. This pattern correlates with and depends on ceramide synthase 3 (CerS3) localization in both, pachytene spermatocytes until completion of meiosis and elongating spermatids. (2) Blocking the pathways to germ cell-specific ceramides (CerS3-KO) and further to glycosphingolipids (glucosylceramide synthase-KO) in mice highlights the need for special SLs for spermatid ICB stability. In contrast to somatic mitosis these SLs require ultra-long polyunsaturated anchors with unique physico-chemical properties, which can only be provided by CerS3. Loss of these anchors causes enhanced apoptosis during meiosis, formation of multinuclear giant cells and spermatogenic arrest. Hence, testis-specific SLs, which we also link to CerS3 in human testis, are quintessential for male fertility.

Description: Therapy-responsive biomarkers are an important and unmet need in the muscular dystrophy field where new treatments are currently in clinical trials. By using a comprehensive high-resolution mass spectrometry approach and western blot validation, we found that two fragments of the myofibrillar structural protein myomesin-3 (MYOM3) are abnormally present in sera of Duchenne muscular dystrophy (DMD) patients, limb-girdle muscular dystrophy type 2D (LGMD2D) and their respective animal models. Levels of MYOM3 fragments were assayed in therapeutic model systems: (1) restoration of dystrophin expression by antisense oligonucleotide-mediated exon-skipping in mdx mice and (2) stable restoration of α-sarcoglycan expression in KO-SGCA mice by systemic injection of a viral vector. Following administration of the therapeutic agents MYOM3 was restored toward wild-type levels. In the LGMD model, where different doses of vector were used, MYOM3 restoration was dose-dependent. MYOM3 fragments showed lower inter-individual variability compared with the commonly used creatine kinase assay, and correlated better with the restoration of the dystrophin-associated protein complex and muscle force. These data suggest that the MYOM3 fragments hold promise for minimally invasive assessment of experimental therapies for DMD and other neuromuscular disorders.

Description: RNA interference (RNAi) offers a promising therapeutic approach for dominant genetic disorders that involve gain-of-function mechanisms. One candidate disease for RNAi therapy application is myotonic dystrophy type 1 (DM1), which results from toxicity of a mutant mRNA. DM1 is caused by expansion of a CTG repeat in the 3' UTR of the DMPK gene. The expression of DMPK mRNA containing an expanded CUG repeat (CUG exp ) leads to defects in RNA biogenesis and turnover. We designed miRNA-based RNAi hairpins to target the CUG exp mRNA in the human α-skeletal muscle actin long-repeat ( HSA LR ) mouse model of DM1. RNAi expression cassettes were delivered to HSA LR mice using recombinant adeno-associated viral (rAAV) vectors injected intravenously as a route to systemic gene therapy. Vector delivery significantly reduced disease pathology in muscles of the HSA LR mice, including a reduction in the CUG exp mRNA, a reduction in myotonic discharges, a shift toward adult pre-mRNA splicing patterns, reduced myofiber hypertrophy and a decrease in myonuclear foci containing the CUG exp mRNA. Significant reversal of hallmarks of DM1 in the rAAV RNAi-treated HSA LR mice indicate that defects characteristic of DM1 can be mitigated with a systemic RNAi approach targeting the nuclei of terminally differentiated myofibers. Efficient rAAV-mediated delivery of RNAi has the potential to provide a long-term therapy for DM1 and other dominant muscular dystrophies.

Description: The heart is a muscle with high energy demands. Hence, most patients with mitochondrial disease produced by defects in the oxidative phosphorylation (OXPHOS) system are susceptible to cardiac involvement. The presentation of mitochondrial cardiomyopathy includes hypertrophic, dilated and left ventricular noncompaction, but the molecular mechanisms involved in cardiac impairment are unknown. One of the most frequent OXPHOS defects in humans frequently associated with cardiomyopathy is cytochrome c oxidase (COX) deficiency caused by mutations in COX assembly factors such as Sco1 and Sco2. To investigate the molecular mechanisms that underlie the cardiomyopathy associated with Sco deficiency, we have heart specifically interfered scox expression, the single Drosophila Sco orthologue. Cardiac-specific knockdown of scox reduces fly lifespan, and it severely compromises heart function and structure, producing dilated cardiomyopathy. Cardiomyocytes with low levels of scox have a significant reduction in COX activity and they undergo a metabolic switch from OXPHOS to glycolysis, mimicking the clinical features found in patients harbouring Sco mutations. The major cardiac defects observed are produced by a significant increase in apoptosis, which is dp53-dependent. Genetic and molecular evidence strongly suggest that dp53 is directly involved in the development of the cardiomyopathy induced by scox deficiency. Remarkably, apoptosis is enhanced in the muscle and liver of Sco2 knock-out mice, clearly suggesting that cell death is a key feature of the COX deficiencies produced by mutations in Sco genes in humans.

Description: Understanding the regulatory landscape of the human genome is a central question in complex trait genetics. Most single-nucleotide polymorphisms (SNPs) associated with cancer risk lie in non-protein-coding regions, implicating regulatory DNA elements as functional targets of susceptibility variants. Here, we describe genome-wide annotation of regions of open chromatin and histone modification in fallopian tube and ovarian surface epithelial cells (FTSECs, OSECs), the debated cellular origins of high-grade serous ovarian cancers (HGSOCs) and in endometriosis epithelial cells (EECs), the likely precursor of clear cell ovarian carcinomas (CCOCs). The regulatory architecture of these cell types was compared with normal human mammary epithelial cells and LNCaP prostate cancer cells. We observed similar positional patterns of global enhancer signatures across the three different ovarian cancer precursor cell types, and evidence of tissue-specific regulatory signatures compared to non-gynecological cell types. We found significant enrichment for risk-associated SNPs intersecting regulatory biofeatures at 17 known HGSOC susceptibility loci in FTSECs ( P = 3.8 x 10 –30 ), OSECs ( P = 2.4 x 10 –23 ) and HMECs ( P = 6.7 x 10 –15 ) but not for EECs ( P = 0.45) or LNCaP cells ( P = 0.88). Hierarchical clustering of risk SNPs conditioned on the six different cell types indicates FTSECs and OSECs are highly related (96% of samples using multi-scale bootstrapping) suggesting both cell types may be precursors of HGSOC. These data represent the first description of regulatory catalogues of normal precursor cells for different ovarian cancer subtypes, and provide unique insights into the tissue specific regulatory variation with respect to the likely functional targets of germline genetic susceptibility variants for ovarian cancer.

Description: The gene mapt codes for the microtubule-associated protein Tau. The R406W amino acid substitution in Tau is associated with frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) characterized by Tau-positive filamentous inclusions. These filamentous Tau inclusions are present in a group of neurodegenerative diseases known as tauopathies, including Alzheimer's disease (AD). To gain more insights into the pathomechanism of tauopathies, we performed an RNAi-based large-scale screen in Drosophila melanogaster to identify genetic modifiers of Tau[R406W]-induced toxicity. A collection of RNAi lines, putatively silencing more than 7000 genes, was screened for the ability to modify Tau[R406W]-induced toxicity in vivo . This collection covered more than 50% of all protein coding fly genes and more than 90% of all fly genes known to have a human ortholog. Hereby, we identified 62 genes that, when silenced by RNAi, modified Tau-induced toxicity specifically. Among these 62 modifiers were three subunits of the Dynein/Dynactin complex. Analysis on segmental nerves of fly larvae showed that pan neural Tau[R406W] expression and concomitant silencing of Dynein/Dynactin complex members synergistically caused strong pathological changes within the axonal compartment, but only minor changes at synapses. At the larval stage, these alterations did not cause locomotion deficits, but became evident in adult flies. Our data suggest that Tau-induced detrimental effects most likely originate from axonal rather than synaptic dysfunction and that impaired retrograde transport intensifies detrimental effects of Tau in axons. In conclusion, our findings contribute to the elucidation of disease mechanisms in tauopathies like FTDP-17 or AD.

Description: Retinitis pigmentosa (RP), the most common form of inherited retinal degeneration, is clinically and genetically heterogeneous and can appear as syndromic or non-syndromic. Mucopolysaccharidosis type IIIC (MPS IIIC) is a lethal disorder, caused by mutations in the heparan-alpha-glucosaminide N-acetyltransferase ( HGSNAT ) gene and characterized by progressive neurological deterioration, with retinal degeneration as a prominent feature. We identified HGSNAT mutations in six patients with non-syndromic RP. Whole exome sequencing (WES) in an Ashkenazi Jewish Israeli RP patient revealed a novel homozygous HGSNAT variant, c.370A〉T, which leads to partial skipping of exon 3. Screening of 66 Ashkenazi RP index cases revealed an additional family with two siblings homozygous for c.370A〉T. WES in three Dutch siblings with RP revealed a complex HGSNAT variant, c.[398G〉C; 1843G〉A] on one allele, and c.1843G〉A on the other allele. HGSNAT activity levels in blood leukocytes of patients were reduced compared with healthy controls, but usually higher than those in MPS IIIC patients. All patients were diagnosed with non-syndromic RP and did not exhibit neurological deterioration, or any phenotypic features consistent with MPS IIIC. Furthermore, four of the patients were over 60 years old, exceeding by far the life expectancy of MPS IIIC patients. HGSNAT is highly expressed in the mouse retina, and we hypothesize that the retina requires higher HGSNAT activity to maintain proper function, compared with other tissues associated with MPS IIIC, such as the brain. This report broadens the spectrum of phenotypes associated with HGSNAT mutations and highlights the critical function of HGSNAT in the human retina.

Description: Increased age, BMI and HbA1c levels are risk factors for several non-communicable diseases. However, the impact of these factors on the genome-wide DNA methylation pattern in human adipose tissue remains unknown. We analyzed the DNA methylation of ~480 000 sites in human adipose tissue from 96 males and 94 females and related methylation to age, BMI and HbA1c. We also compared epigenetic signatures in adipose tissue and blood. Age was significantly associated with both altered DNA methylation and expression of 1050 genes (e.g. FHL2 , NOX4 and PLG ). Interestingly, many reported epigenetic biomarkers of aging in blood, including ELOVL2 , FHL2 , KLF14 and GLRA1 , also showed significant correlations between adipose tissue DNA methylation and age in our study. The most significant association between age and adipose tissue DNA methylation was found upstream of ELOVL2 . We identified 2825 genes (e.g. FTO , ITIH5 , CCL18 , MTCH2 , IRS1 and SPP1 ) where both DNA methylation and expression correlated with BMI. Methylation at previously reported HIF3A sites correlated significantly with BMI in females only. HbA1c (range 28–46 mmol/mol) correlated significantly with the methylation of 711 sites, annotated to, for example, RAB37 , TICAM1 and HLA-DPB1 . Pathway analyses demonstrated that methylation levels associated with age and BMI are overrepresented among genes involved in cancer, type 2 diabetes and cardiovascular disease. Our results highlight the impact of age, BMI and HbA1c on epigenetic variation of candidate genes for obesity, type 2 diabetes and cancer in human adipose tissue. Importantly, we demonstrate that epigenetic biomarkers in blood can mirror age-related epigenetic signatures in target tissues for metabolic diseases such as adipose tissue.

Description: Interstitial lung disease, nephrotic syndrome and junctional epidermolysis bullosa is an autosomal recessive multiorgan disorder caused by mutations in the gene for the integrin α3 subunit ( ITGA3 ). The full spectrum of manifestations and genotype–phenotype correlations is still poorly characterized. Here, we uncovered the disease-causing role and the molecular mechanisms underlying a homozygous ITGA3 mutation leading to the single amino acid substitution, p.R463W. The patient suffered from respiratory distress and episodes of cyanosis with onset in the first week of life and had a nephrotic syndrome. Although there was no clinical evidence for cutaneous fragility, the analysis of a skin sample and of skin epithelial cells enabled the direct assessment of the authentic mutant protein. We show that the mutation altered the conformation of the extracellular β-propeller domain of the integrin α3 subunit preventing correct processing of N-linked oligosaccharides, heterodimerization with β1 integrin and maturation through cleavage into heavy and light chains in the Golgi. Confocal microscopy demonstrated that the mutant protein accumulated intracellularly, but it was not present in focal adhesions or on the cell membrane as shown by flow cytometry. These findings highlight that single amino acid changes in the integrin α3 subunit may crucially alter the structure and complex processing of this integrin, completely preventing its functionality. The present report also underscores that ITGA3 mutations may account for atypical cases solely with early onset respiratory and renal involvement.

Description: Gestational age (GA) and birth weight have been implicated in the determination of long-term health. It has been hypothesized that changes in DNA methylation may mediate these long-term effects. We obtained DNA methylation profiles from cord blood and peripheral blood at ages 7 and 17 in the same children from the Avon Longitudinal Study of Parents and Children. Repeated-measures data were used to investigate changes in birth-related methylation during childhood and adolescence. Ten developmental phenotypes (e.g. height) were analysed to identify possible mediation of health effects by DNA methylation. In cord blood, methylation at 224 CpG sites was found to be associated with GA and 23 CpG sites with birth weight. Methylation changed in the majority of these sites over time, but neither birth characteristic was strongly associated with methylation at age 7 or 17 (using a conservative correction for multiple testing of P 〈 1.03 x 10 –7 ), suggesting resolution of differential methylation by early childhood. Associations were observed between birth weight-associated CpG sites and phenotypic characteristics in childhood. One strong association involved birth weight, methylation of a CpG site proximal to the NFIX locus and bone mineral density at age 17. Analysis of serial methylation from birth to adolescence provided evidence for a lack of persistence of methylation differences beyond early childhood. Sites associated with birth weight were linked to developmental genes and have methylation levels which are associated with developmental phenotypes. Replication and interrogation of causal relationships are needed to substantiate whether methylation differences at birth influence the association between birth weight and development.

Description: Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset neurodegenerative disorder affecting carriers of the fragile X-premutation, who have an expanded CGG repeat in the 5'-UTR of the FMR1 gene. FXTAS is characterized by progressive development of intention tremor, ataxia, parkinsonism and neuropsychological problems. The disease is thought to be caused by a toxic RNA gain-of-function mechanism, and the major hallmark of the disease is ubiquitin-positive intranuclear inclusions in neurons and astrocytes. We have developed a new transgenic mouse model in which we can induce expression of an expanded repeat in the brain upon doxycycline (dox) exposure (i.e. Tet-On mice). This Tet-On model makes use of the PrP-rtTA driver and allows us to study disease progression and possibilities of reversibility. In these mice, 8 weeks of dox exposure was sufficient to induce the formation of ubiquitin-positive intranuclear inclusions, which also stain positive for the RAN translation product FMRpolyG. Formation of these inclusions is reversible after stopping expression of the expanded CGG RNA at an early developmental stage. Furthermore, we observed a deficit in the compensatory eye movements of mice with inclusions, a functional phenotype that could be reduced by stopping expression of the expanded CGG RNA early in the disease development. Taken together, this study shows, for the first time, the potential of disease reversibility and suggests that early intervention might be beneficial for FXTAS patients.

Description: Mutations affecting specific splicing regulatory elements offer suitable models to better understand their interplay and to devise therapeutic strategies. Here we characterize a meaningful splicing model in which numerous Hemophilia B-causing mutations, either missense or at the donor splice site (5'ss) of coagulation F9 exon 2, promote aberrant splicing by inducing the usage of a strong exonic cryptic 5'ss. Splicing assays with natural and artificial F9 variants indicated that the cryptic 5'ss is regulated, among a network of regulatory elements, by an exonic splicing silencer (ESS). This finding and the comparative analysis of the F9 sequence across species showing that the cryptic 5'ss is always paralleled by the conserved ESS support a compensatory mechanism aimed at minimizing unproductive splicing. To recover splicing we tested antisense oligoribonucleotides masking the cryptic 5'ss, which were effective on exonic changes but promoted exon 2 skipping in the presence of mutations at the authentic 5'ss. On the other hand, we observed a very poor correction effect by small nuclear RNA U1 (U1snRNA) variants with increased or perfect complementarity to the defective 5'ss, a strategy previously exploited to rescue splicing. Noticeably, the combination of the mutant-specific U1snRNAs with antisense oligonucleotides produced appreciable amounts of correctly spliced transcripts (from 0 to 20–40%) from several mutants of the exon 2 5'ss. Based on the evidence of an altered interplay among ESS, cryptic and the authentic 5'ss as a disease-causing mechanism, we provide novel experimental insights into the combinatorial correction activity of antisense molecules and compensatory U1snRNAs.

Description: Facioscapulohumeral muscular dystrophy (FSHD) is caused by the aberrant expression of the DUX4 transcription factor in skeletal muscle. The DUX4 retrogene is encoded in the D4Z4 macrosatellite repeat array, and smaller array size or a mutation in the SMCHD1 gene results in inefficient epigenetic repression of DUX4 in skeletal muscle, causing FSHD1 and FSHD2, respectively. Previously we showed that the entire D4Z4 repeat is bi-directionally transcribed with the generation of small si- or miRNA-like fragments and suggested that these might suppress DUX4 expression through the endogenous RNAi pathway. Here we show that exogenous siRNA targeting the region upstream of the DUX4 transcription start site suppressed DUX4 mRNA expression and increased both H3K9 methylation and AGO2 recruitment. In contrast, similarly targeted MOE-gapmer antisense oligonucleotides that degrade RNA but do not engage the RNAi pathway did not repress DUX4 expression. In addition, knockdown of DICER or AGO2 using either siRNA or MOE-gapmer chemistries resulted in the induction of DUX4 expression in control muscle cells that normally do not express DUX4 , indicating that the endogenous RNAi pathway is necessary to maintain repression of DUX4 in control muscle cells. Together these data demonstrate a role of the endogenous RNAi pathway in repeat-mediated epigenetic repression of the D4Z4 macrosatellite repeat, and show that enhancing the activity of this pathway by supplying exogenous siRNA oligonucleotides represents a potential therapeutic approach to silencing DUX4 in FSHD.

Description: Overgrowth syndromes comprise a group of heterogeneous disorders characterised by excessive growth parameters, often in association with intellectual disability. To identify new causes of human overgrowth, we have been undertaking trio-based exome sequencing studies in overgrowth patients and their unaffected parents. Prioritisation of functionally relevant genes with multiple unique de novo mutations revealed four mutations in protein phosphatase 2A (PP2A) regulatory subunit B family genes protein phosphatase 2, regulatory Subunit B’, beta (PPP2R5B) ; protein phosphatase 2, regulatory Subunit B’, gamma (PPP2R5C) ; and protein phosphatase 2, regulatory Subunit B’, delta (PPP2R5D). This observation in 3 related genes in 111 individuals with a similar phenotype is greatly in excess of the expected number, as determined from gene-specific de novo mutation rates ( P = 1.43 x 10 –10 ). Analysis of exome-sequencing data from a follow-up series of overgrowth probands identified a further pathogenic mutation, bringing the total number of affected individuals to 5. Heterozygotes shared similar phenotypic features including increased height, increased head circumference and intellectual disability. The mutations clustered within a region of nine amino acid residues in the aligned protein sequences ( P = 1.6 x 10 –5 ). We mapped the mutations onto the crystal structure of the PP2A holoenzyme complex to predict their molecular and functional consequences. These studies suggest that the mutations may affect substrate binding, thus perturbing the ability of PP2A to dephosphorylate particular protein substrates. PP2A is a major negative regulator of v-akt murine thymoma viral oncogene homolog 1 (AKT). Thus, our data further expand the list of genes encoding components of the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/AKT signalling cascade that are disrupted in human overgrowth conditions.

Description: Miles–Carpenter syndrome (MCS) was described in 1991 as an XLID syndrome with fingertip arches and contractures and mapped to proximal Xq. Patients had microcephaly, short stature, mild spasticity, thoracic scoliosis, hyperextendable MCP joints, rocker-bottom feet, hyperextended elbows and knees. A mutation, p.L66H, in ZC4H2 , was identified in a XLID re-sequencing project. Additional screening of linked families and next generation sequencing of XLID families identified three ZC4H2 mutations: p.R18K, p.R213W and p.V75in15aa. The families shared some relevant clinical features. In silico modeling of the mutant proteins indicated all alterations would destabilize the protein. Knockout mutations in zc4h2 were created in zebrafish and homozygous mutant larvae exhibited abnormal swimming, increased twitching, defective eye movement and pectoral fin contractures. Because several of the behavioral defects were consistent with hyperactivity, we examined the underlying neuronal defects and found that sensory neurons and motoneurons appeared normal. However, we observed a striking reduction in GABAergic interneurons. Analysis of cell-type-specific markers showed a specific loss of V2 interneurons in the brain and spinal cord, likely arising from mis-specification of neural progenitors. Injected human wt ZC4H2 rescued the mutant phenotype. Mutant zebrafish injected with human p.L66H or p.R213W mRNA failed to be rescued, while the p.R18K mRNA was able to rescue the interneuron defect. Our findings clearly support ZC4H2 as a novel XLID gene with a required function in interneuron development. Loss of function of ZC4H2 thus likely results in altered connectivity of many brain and spinal circuits.

Description: Alterations in oxidative metabolism are considered to be one of the major contributors to Huntington's disease (HD) pathogenesis. However, existing data about oxidative metabolism in HD are contradictory. Here, we investigated the effect of mutant huntingtin (mHtt) on oxidative metabolism in YAC128 mice. Both mHtt and wild-type huntingtin (Htt) were associated with mitochondria and the amount of bound Htt was four-times higher than the amount of bound mHtt. Percoll gradient-purified brain synaptic and non-synaptic mitochondria as well as unpurified brain, liver and heart mitochondria, isolated from 2- and 10-month-old YAC128 mice and age-matched WT littermates had similar respiratory rates. There was no difference in mitochondrial membrane potential or ADP and ATP levels. Expression of selected nuclear-encoded mitochondrial proteins in 2- and 10-month-old YAC128 and WT mice was similar. Cultured striatal and cortical neurons from YAC128 and WT mice had similar respiratory and glycolytic activities as measured with Seahorse XF24 analyzer in medium containing 10 m m glucose and 15 m m pyruvate. In the medium with 2.5 m m glucose, YAC128 striatal neurons had similar respiration, but slightly lower glycolytic activity. Striatal neurons had lower maximal respiration compared with cortical neurons. In vivo experiments with YAC128 and WT mice showed similar O 2 consumption, CO 2 release, physical activity, food consumption and fasted blood glucose. However, YAC128 mice were heavier and had more body fat compared with WT mice. Overall, our data argue against respiratory deficiency in YAC128 mice and, consequently, suggest that mitochondrial respiratory dysfunction is not essential for HD pathogenesis.

Description: Leucine-rich repeat kinase 2 (LRRK2) is the causative molecule of the autosomal dominant hereditary form of Parkinson's disease (PD), PARK8, which was originally defined in a study of a Japanese family (the Sagamihara family) harboring the I2020T mutation in the kinase domain. Although a number of reported studies have focused on cell death mediated by mutant LRRK2, details of the pathogenetic effect of LRRK2 still remain to be elucidated. In the present study, to elucidate the mechanism of neurodegeneration in PD caused by LRRK2, we generated induced pluripotent stem cells (iPSC) derived from fibroblasts of PD patients with I2020T LRRK2 in the Sagamihara family. We found that I2020T mutant LRRK2 iPSC-derived neurons released less dopamine than control-iPSC-derived neurons. Furthermore, we demonstrated that patient iPSC-derived neurons had a lower phospho-AKT level than control-iPSC-derived neurons, and that the former showed an increased incidence of apoptosis relative to the controls. Interestingly, patient iPSC-derived neurons exhibited activation of glycogen synthase kinase-3β (GSK-3β) and high Tau phosphorylation. In addition, the postmortem brain of the patient from whom the iPSC had been established exhibited deposition of neurofibrillary tangles as well as increased Tau phosphorylation in neurons. These results suggest that I2020T LRRK2-iPSC could be a promising new tool for reproducing the pathology of PD in the brain caused by the I2020T mutation, and applicable as a model in studies of targeted therapeutics.

Description: SOX10 is a transcription factor with well-known functions in neural crest and oligodendrocyte development. Mutations in SOX10 were first associated with Waardenburg–Hirschsprung disease (WS4; deafness, pigmentation defects and intestinal aganglionosis). However, variable phenotypes that extend beyond the WS4 definition are now reported. The neurological phenotypes associated with some truncating mutations are suggested to be the result of escape from the nonsense-mediated mRNA decay pathway; but, to date, no mechanism has been suggested for missense mutations, of which approximately 20 have now been reported, with about half of the latter shown to be redistributed to nuclear bodies of undetermined nature and function in vitro . Here, we report that p54NRB, which plays a crucial role in the regulation of gene expression during many cellular processes including differentiation, interacts synergistically with SOX10 to regulate several target genes. Interestingly, this paraspeckle protein, as well as two other members of the Drosophila behavior human splicing (DBHS) protein family, co-localize with SOX10 mutants in nuclear bodies, suggesting the possible paraspeckle nature of these foci or re-localization of the DBHS members to other subnuclear compartments. Remarkably, the co-transfection of wild-type and mutant SOX10 constructs led to the sequestration of wild-type protein in mutant-induced foci. In contrast to mutants presenting with additional cytoplasmic re-localization, those exclusively found in the nucleus alter synergistic activity between SOX10 and p54NRB. We propose that such a dominant negative effect may contribute to or be at the origin of the unique progressive and severe neurological phenotype observed in affected patients.

Description: Huntington's disease (HD) is a hereditary neurodegenerative disorder characterized by motor and cognitive impairments, involving striatum, cortex and hippocampus. Synaptic and memory dysfunction in HD mouse models have been related to low levels of brain-derived neurotrophic factor (BDNF) and imbalance between TrkB and p75 NTR receptors. In addition, astrocyte over-activation has also been suggested to contribute to HD cognitive deficits. Fingolimod (FTY720), a modulator of sphingosine-1 phosphate (S1P) receptors, has been shown to increase BDNF levels and to reduce astrogliosis, proving its potential to regulate trophic support and inflammatory response. In this view, we have investigated whether FTY720 improves synaptic plasticity and memory in the R6/1 mouse model of HD, through regulation of BDNF signaling and astroglial reactivity. Chronic administration of FTY720 from pre-symptomatic stages ameliorated long-term memory deficits and dendritic spine loss in CA1 hippocampal neurons from R6/1 mice. Furthermore, FTY720 delivery prevented astrogliosis and over-activation of nuclear factor kappa beta (NF-B) signaling in the R6/1 hippocampus, reducing tumor necrosis factor alpha (TNFα) and induced nitric oxide synthase (iNOS) levels. TNFα decrease correlated with the normalization of p75 NTR expression in the hippocampus of FTY720-treated R6/1 mice, thus preventing p75 NTR /TrkB imbalance. In addition, FTY720 increased cAMP levels and promoted phosphorylation of CREB and RhoA in the hippocampus of R6/1 mice, further supporting its role in the enhancement of synaptic plasticity. Our findings provide new insights into the mechanism of action of FTY720 and reveal a novel therapeutic strategy to treat memory deficits in HD.

Description: DDX11 was recently identified as a cause of Warsaw breakage syndrome (WABS). However, the functional mechanism of DDX11 and the contribution of clinically described mutations to the pathogenesis of WABS are elusive. Here, we show that DDX11 is a novel nucleolar protein that preferentially binds to hypomethylated active ribosomal DNA (rDNA) gene loci, where it interacts with upstream binding factor (UBF) and the RNA polymerase I (Pol I). DDX11 knockdown changed the epigenetic state of rDNA loci from euchromatic structures to more heterochromatic structures, reduced the activity of UBF, decreased the recruitment of UBF and RPA194 (a subunit of Pol I) to rDNA promoter, suppressed rRNA transcription and thereby inhibited growth and proliferation of HeLa cells. Importantly, two indentified WABS-derived mutants, R263Q and K897del, and a Fe–S deletion construct demonstrated significantly reduced binding abilities to rDNA promoters and lowered DNA-dependent ATPase activities compared with wild-type DDX11. Knockdown of the zebrafish ortholog of human DDX11 by morpholinos resulted in growth retardation and vertebral and craniofacial malformations in zebrafish, concomitant with the changes in histone epigenetic modifications at rDNA loci, the reduction of Pol I recruitment to the rDNA promoter and a significant decrease in nascent pre-RNA levels. These growth disruptions in zebrafish in response to DDX11 reduction showed similarities to the clinically described developmental abnormalities found in WABS patients for the first time in any vertebrate. Thus, our results indicate that DDX11 functions as a positive regulator of rRNA transcription and provides a novel insight into the pathogenesis of WABS.

Description: Genome-wide association studies (GWAS) have identified several common loci contributing to non-obstructive azoospermia (NOA). However, a substantial fraction of NOA heritability remains undefined, especially those low-frequency [defined here as having a minor allele frequency (MAF) between 0.5 and 5%] and rare (MAF below 0.5%) variants. Here, we performed a 3-stage exome-wide association study in Han Chinese men to evaluate the role of low-frequency or rare germline variants in NOA development. The discovery stage included 962 NOA cases and 1348 healthy male controls genotyped by exome chips and was followed by a 2-stage replication with an additional 2168 cases and 5248 controls. We identified three low-frequency variants located at 6p22.2 (rs2298090 in HIST1H1E encoding p.Lys152Arg: OR = 0.30, P = 2.40 x 10 –16 ) and 6p21.33 (rs200847762 in FKBPL encoding p.Pro137Leu: OR = 0.11, P = 3.77 x 10 –16 ; rs11754464 in MSH5 : OR = 1.78, P = 3.71 x 10 –7 ) associated with NOA risk after Bonferroni correction. In summary, we report an instance of newly identified signals for NOA risk in genes previously undetected through GWAS on 6p22.2–6p21.33 in a Chinese population and highlight the role of low-frequency variants with a large effect in the process of spermatogenesis.

Description: Loss-of-function mutations in the X-linked gene Methyl-CpG-binding protein 2 ( MECP2 ) cause a devastating pediatric neurological disorder called Rett syndrome. In males, these mutations typically result in severe neonatal encephalopathy and early lethality. On the other hand, owing to expression of the normal allele in ~50% of cells, females do not suffer encephalopathy but instead develop Rett syndrome. Typically females with Rett syndrome exhibit a delayed onset of neurologic dysfunction that manifests around the child's first birthday and progresses over the next few years. Features of this disorder include loss of acquired language and motor skills, intellectual impairment and hand stereotypies. The developmental regression observed in patients with Rett syndrome arises from altered neuronal function and is not the result of neurodegeneration. Maintenance of an appropriate level of MeCP2 appears integral to the function of healthy neurons as patients with increased levels of MeCP2, owing to duplication of the Xq28 region encompassing the MECP2 locus, also present with intellectual disability and progressive neurologic symptoms. Despite major efforts over the past two decades to elucidate the molecular functions of MeCP2, the mechanisms underlying the delayed appearance of symptoms remain unclear. In this review, we will highlight recent findings that have expanded our knowledge of MeCP2's functions, and we will discuss how epigenetic regulation, chromatin organization and circuit dynamics may contribute to the postnatal onset of Rett syndrome.

Description: The Matachewan Large Igneous Province (LIP) is interpreted to have formed during the early stages of mantle plume-induced continental break-up in the early Proterozoic. When the Matachewan LIP is reconstructed to its original configuration with units from the Superior Craton and other formerly adjacent blocks (Karelia, Kola, Wyoming and Hearne), the dyke swarms, layered intrusions and flood basalts, emplaced over the lifetime of the province, form one of the most extensive magmatic provinces recognized in the geological record. New geochemical data allow, for the first time, the Matachewan LIP to be considered as a single, coherent entity and show that Matachewan LIP rocks share a common tholeiitic composition and trace element geochemistry, characterized by enrichment in the most incompatible elements and depletion in the less incompatible elements. This signature, ubiquitous in early Proterozoic continental magmatic rocks, may indicate that the Matachewan LIP formed through contamination of the primary magmas with lithospheric material or that the early Proterozoic mantle had a fundamentally different composition from the modern mantle. In addition to the radiating geometry of the dyke swarms, a plume origin for the Matachewan LIP is consistent with the geochemistry of some of the suites; these suites are used to constrain a source mantle potential temperature of c. 1500–1550°C. Comparison of these mantle potential temperatures with estimated temperatures for the early Proterozoic upper mantle indicates that they are consistent with a hot mantle plume source for the magmatism. Geochemical data from coeval intrusions suggest that the plume head was compositionally heterogeneous and sampled material from both depleted and enriched mantle. As has been documented with less ancient but similarly vast LIPs, the emplacement of the Matachewan LIP probably had a significant impact on the early Proterozoic global environment. Compilation of the best age estimates for various suites shows that the emplacement of the Matachewan LIP occurred synchronously with the Great Oxidation Event. We explore the potential for the eruption of this LIP and the emission of its associated volcanic gases to have been a driver of the irreversible oxygenation of the Earth.

Description: Olivine major and trace element compositions from 12 basalts from the southern Payenia volcanic province in Argentina have been analyzed by electron microprobe and laser ablation inductively coupled plasma mass spectrometry. The olivines have high Fe/Mn and low Ca/Fe and many fall at the end of the global olivine array, indicating that they were formed from a pyroxene-rich source distinct from typical mantle peridotite. The olivines with the highest Fe/Mn have higher Zn/Fe, Zn and Co and lower Co/Fe than the olivines with lower Fe/Mn, also suggesting contributions from a pyroxene-rich source. Together with whole-rock radiogenic isotopes and elemental concentrations, the samples indicate mixing between two mantle sources: (1) a pyroxene-rich source with EM-1 ocean island basalt type trace element and isotope characteristics; (2) a peridotitic source with more radiogenic Pb that was metasomatized by subduction-zone fluids and/or melts. The increasing contributions from the pyroxene-rich source in the southern Payenia basalts are correlated with an increasing Fe-enrichment, which caused the olivines to have lower forsterite contents at a given Ni content. Al-in-olivine crystallization temperatures measured on olivine–spinel pairs are between 1155 and 1243°C and indicate that the magmas formed at normal upper mantle (asthenospheric) temperatures of ~1350°C. The pyroxene-rich material is interpreted to have been brought up from the deeper parts of the upper mantle by vigorous asthenospheric upwelling caused by break-off of the Nazca slab south of Payenia during the Pliocene and roll-back of the subducting slab beneath Payenia. The pyroxene-rich mantle mixed with peridotitic metasomatized South Atlantic mantle in the mantle wedge beneath Payenia.

Description: To reconstruct the magmatic–hydrothermal processes leading to porphyry Mo ore formation at the Climax Mo mine, Colorado, four magma units that were emplaced before, during and shortly after the mineralization events were investigated: (1) a pre-mineralization white dike of the Alma district; (2) the syn-mineralization Chalk Mountain Rhyolite; (3) a late- to post-mineralization rhyolite porphyry dyke; (4) a mafic enclave within the productive Bartlett stock. Melt inclusions, mineral inclusions and fluid inclusions in quartz phenocrysts were investigated by means of laser ablation inductively coupled plasma mass spectrometry, electron microprobe and microthermometry. Based on melt inclusion data both the Chalk Mountain Rhyolite and the rhyolite porphyry were ~10 times more fractionated than average granite and show geochemical characteristics of topaz rhyolites. They were saturated in magnetite, Mn-rich ilmenite, fluorite, aeschynite, monazite, pyrrhotite and thorite, and crystallized predominantly at 710–730°C, 1·2–2·6 kbar and log f O 2 FMQ + 2·2 (where FMQ is fayalite–magnetite–quartz). The silicate melt of the Chalk Mountain Rhyolite contained 3·5 ± 0·4 wt % F, 0·09 ± 0·03 wt % Cl, ≥ 3·0 wt % H 2 O, 15–90 µg g –1 Cs, 500–1500 µg g –1 Rb and 5–7 µg g –1 Mo, whereas that of the rhyolite porphyry contained 1·1 ± 0·3 wt % F and 4·9 ± 1·2 wt % H 2 O, but otherwise had a virtually identical major and trace element composition. The fluid exsolving from the latter melt had a bulk salinity of 10 ± 2 wt % NaCl equiv and contained of the order of 100 µg g –1 Mo. After emplacement of the Chalk Mountain Rhyolite magma at subvolcanic levels, extremely fractionated silicate melts coexisting with hypersaline brines (salt melts) and low-density vapor percolated at near-solidus conditions through the rock. These silicate melts contained 6·6 ± 0·4 wt % F, ≥ 7·5 ± 0·6 wt % H 2 O, 0·51 ± 0·05 wt % Cl, and up to 0·5 wt % Cs and 100 µg g –1 Mo, whereas the hypersaline brines contained 1–2 wt % Cs and 0·3–0·6 wt % Mo. However, owing to their negligible masses these liquids are unlikely to have played a major role in the mineralization process. The majority of Mo in the Climax deposit appears to have been derived from melts containing 5–7 µg g –1 Mo and bulk fluids containing ~100 µg g –1 Mo. These concentrations are similar to those found in similarly fractionated melts and fluids in barren and sub-economically mineralized intrusions. However, whereas in the latter intrusions fractionated melts occurred in a rather dispersed state, they seem to have been present as large, coherent masses in the apical parts of Climax-type porphyry Mo-forming magma systems. Efficient segregation of fractionated melts and fluids into the top of mineralizing magma chambers appears to have been promoted by high fluorine concentrations in the silicate melt, which was partly a primary feature, and partly an indirect consequence of other characteristics of within-plate magmatism.

Description: The Skaergaard intrusion, Greenland, is the type locality for Skaergaard-type mineralizations. Mineralization levels are perfectly concordant with igneous layering, up to 5 m thick, internally fractionated, and contain crystallized sulphide droplets and precious metal alloys, sulphides, arsenides and telluride. Immiscible Cu-rich sulphide droplets, formed in a mush zone below the roof, scavenged precious metals. They were subsequently dissolved and transported to the floor in late-formed, immiscible, Fe-rich mush melts. Mineralized stratigraphic intervals of floor gabbro formed in ‘proto-macrolayers‘, owing to local sulphide saturation in melt concentrated between floating plagioclase and sinking clinopyroxene. The floor mineralization is divided into four stratigraphic sections. Formation of the Lower Platinum Group Element Mineralization (LPGEM) involved: (1) crystallization of the bulk liquid liquidus paragenesis and in situ fractionation; (2) sulphide saturation and formation of sulphide droplets in melt in the upper part of ‘proto-macrolayers‘. After further in situ fractionation, the following steps occurred: (3) the onset of silicate–silicate immiscibility and the consequent loss of buoyant and immiscible Si-rich melt; (4) dissolution of unprotected droplets of sulphide melt present in the Fe-rich mush melt; (5) compaction-driven upwards loss of residual mush melt enriched in, for example, Au. The LPGEM preserves upward increasing bulk Pd/Pt (~6–13) owing to a continued supply of PGE and Au, with high Pd/Pt. The further development of the LPGEM ceased as the supply of precious metals to the floor waned. The Upper PGE Mineralization (UPGEM) subsequently formed from precious metals recycled in the floor. The UPGEM is characterized by increasing Au substitution in PGE phases, and a decrease in total PGE and Pd/Pt owing to upward fractionation in migrating mush melts and exhaustion of Pd and Pt. An upper Au-rich mineralization level (UAuM) was caused by late remobilization of Au and deposition on grain boundaries in fully crystallized gabbro. Cu concentrations (~150 ppm) are not correlated with PGE and Au. Repeated Cu mineralization levels (CuM), attaining 〉1000 ppm, occur above the Au levels, caused by local mush layer sulphide saturation. PGE, Au and Cu distributions in the floor mineralization reflect sub-liquidus, but supra-solidus, processes and reactions in mushes at the roof, wall and floor. Constraints provided by a new model for the mineralization provide the basis for re-evaluation of the solidification processes in the Skaergaard intrusion. We have identified the importance of extensive in situ fractionation and intrusion-wide elemental redistributions in immiscible Fe- and Si-rich silicate melts. Our model characterizes the floor cumulates as bulk liquid orthocumulates containing an upwards-increasing proportion crystallized from Fe-rich, immiscible mush melt. The roof-rocks are complementary to the floor, with downwards increasing proportions crystallized from the conjugate Si-rich melt. Petrographic observations and the relative timing of crystallization support the hypothesis that crystallization was restricted to marginal mush zones. Bulk melt remaining in the magma chamber evolved not, as generally assumed, as a result of loss of crystals grown from the bulk melt, but as the consequence of mixing with recycled and evolved melt expelled from the mush by compaction. Redistribution of Fe in immiscible melts may be common to mafic intrusions and puts into question the validity of petrogenetic modelling of bulk liquids in mafic intrusions based only on consideration of floor cumulates.

Description: We have determined experimentally the hydrous phase relations and trace element partitioning behaviour of ocean floor basalt protoliths at pressures and temperatures (3 GPa, 750–1000°C) relevant to melting in subduction zones. To avoid potential complexities associated with trace element doping of starting materials we have used natural, pristine mid-ocean ridge basalt (MORB from Kolbeinsey Ridge) and altered oceanic crust (AOC from Deep Sea Drilling Project leg 46, ~20°N Atlantic). Approximately 15 wt % water was added to starting materials to simulate fluid fluxing from dehydrating serpentinite underlying the oceanic crust. The vapour-saturated solidus is sensitive to basalt K 2 O content, decreasing from 825 ± 25°C in MORB (~0·04 wt % K 2 O) to 750°C in AOC (~0·25 wt % K 2 O). Textural evidence indicates that near-solidus fluids are sub-critical in nature. The residual solid assemblage in both MORB and AOC experiments is dominated by garnet and clinopyroxene, with accessory kyanite, epidote, Fe–Ti oxide and rutile (plus quartz–coesite, phengite and apatite below the solidus). Trace element analyses of quenched silica-rich melts show a strong temperature dependence of key trace elements. In contrast to the trace element-doped starting materials of previous studies, we do not observe residual allanite. Instead, abundant residual epidote provides the host for thorium and light rare earth elements (LREE), preventing LREE from being released (LREE 〈3 ppm at 750–900°C). Elevated Ba/Th ratios, characteristic of many arc basalts, are found to be generated within a narrow temperature field above the breakdown temperature of phengite, but below exhaustion of epidote. Melts with Ba/Th 〉1500 and La/Sm PUM (where PUM indicates primitive upper mantle) ~1, most closely matching the geochemical signal of arc lavas worldwide, were generated from AOC at 800–850°C.

Description: We constrain the physical nature of the magma reservoir and the mechanisms of rhyolite generation at Yellowstone caldera via detailed characterization of zircon and sanidine crystals hosted in three rhyolites erupted during the ( c . 170–70 ka) Central Plateau Member eruptive episode—the most recent post-caldera magmatism at Yellowstone. We present 238 U– 230 Th crystallization ages and trace-element compositions of the interiors and surfaces (i.e. unpolished rims) of single zircon crystals from each rhyolite. We compare these zircon data with 238 U– 230 Th crystallization ages of bulk sanidine separates coupled with chemical and isotopic data from single sanidine crystals. Zircon age and trace-element data demonstrate that the magma reservoir that sourced the Central Plateau Member rhyolites was long-lived (150–250 kyr) and genetically related to the preceding episode of magmatism, which occurred c . 256 ka. The interiors of most zircons in each rhyolite were inherited from unerupted material related to older stages of Central Plateau Member magmatism or the preceding late Upper Basin Member magmatism (i.e. are antecrysts). Conversely, most zircon surfaces crystallized near the time of eruption from their host liquids (i.e. are autocrystic). The repeated recycling of zircon interiors from older stages of magmatism demonstrates that sequentially erupted Central Plateau Member rhyolites are genetically related. Sanidine separates from each rhyolite yield 238 U– 230 Th crystallization ages at or near the eruption age of their host magmas, coeval with the coexisting zircon surfaces, but are younger than the coexisting zircon interiors. Chemical and isotopic data from single sanidine crystals demonstrate that the sanidines in each rhyolite are in equilibrium with their host melts, which considered along with their near-eruption crystallization ages suggests that nearly all Central Plateau Member sanidines are autocrystic. The paucity of antecrystic sanidine crystals relative to antecrystic zircons requires a model in which eruptible rhyolites are generated by extracting melt and zircons from a long-lived mush of immobile crystal-rich magma. In this process the larger sanidine crystals remain trapped in the locked crystal network. The extracted melts (plus antecrystic zircon) amalgamate into a liquid-dominated (i.e. eruptible) magma body that is maintained as a physically distinct entity relative to the bulk of the long-lived crystal mush. Zircon surfaces and sanidines in each rhyolite crystallize after melt extraction and amalgamation, and their ages constrain the residence time of eruptible magmas at Yellowstone. Residence times of the large-volume rhyolites (~40–70 km 3 ) are ≤1 kyr (conservatively 〈6 kyr), which suggests that large volumes of rhyolite can be generated rapidly by extracting melt from a crystal mush. Because the lifespan of the crystal mush that sourced the Central Plateau Member rhyolites is two orders of magnitude longer than the residence time of eruptible magma bodies within the reservoir, it is apparent that the Yellowstone magma reservoir spends most of its time in a largely crystalline (i.e. uneruptible) state, similar to the present-day magma reservoir, and that eruptible magma bodies are ephemeral features.

Description: We report and interpret new geochemical and Pb–Sr–Nd isotopic data from 325 samples of shield, late-shield, postshield, and rejuvenated stage lavas from Kauai and Niihau, the two most northwesterly islands in the Hawaiian island chain. Kauai is unique in the Hawaiian chain in that it exhibits a near-continuous geochemical transition from shield to postshield to rejuvenated stage volcanism between 4·4 and 3·6 Ma and has been continuously active over ~6 Myr. From c . 5·7 to 4·3 Ma, the shield stage of both islands produced tholeiitic basalts typical of other Hawaiian shield volcanoes. The Niihau basalts are more evolved and have high Gd/Yb compared with Kauai, indicating a higher residual garnet content in the source. Both Kauai and Niihau shield basalts have Kea-like trace element ratios, but isotopic ratios are transitional between Kea- and Loa-like compositions. The geochemical similarity of the two shields indicates that mantle sources in different regions of the plume source were similar, and that the 〈2 Ma Loa and Kea trends of the southeastern Hawaiian volcanoes are not observed. More Loa-like compositions are evident in shield lavas from eastern Kauai, where the enhanced Loa composition may reflect melting of low-melting temperature plume components as the island migrates off the hotter, more Kea-like, center of the Hawaiian plume. Postshield lavas and intrusive rocks on both islands are rare: Kauai includes alkalic basalts, hawaiites and mugearites that are isotopically homogeneous and include a significant depleted mantle component compared with the shield basalts, whereas the Niihau late-shield and postshield rocks consist of highly contrasting transitional tholeiites or basanites with variable but shield-like isotopic compositions. The Niihau postshield rocks represent variable, but lower degrees of melting of the shield mantle source. Large volumes of rejuvenated stage lavas cover both islands and also form submarine cone fields, but lava compositions are different. On Kauai, rejuvenated lavas range from melilitite to alkalic basalt with trace element, Nd isotope, and Pb isotope ratios that vary as a function of Th and SiO 2 content. Low-degree (high-Th) melts are dominated by a mixed Kea–Loihi component and high-degree (low-Th) melts include more of a depleted rejuvenated component (DRC) typified by rejuvenated stage lavas and xenoliths from nearby Kaula Island. With the exception of a single basanite, the Niihau rejuvenated stage lavas are uniformly alkalic basalt, with Sr and Ba excesses combined with depleted Th and Nb abundances relative to the light rare earth elements. Rejuvenated stage alkalic basalts from both islands are dominated by contributions from the DRC, which have high Sr/Ce and 87 Sr/ 86 Sr but low 206 Pb/ 204 Pb. The Sr-rich, possibly carbonate-bearing, DRC component may be distributed patchily in the rejuvenated stage mantle source such that, where present, the degree of partial melting was enhanced compared with the degree of partial melting of the Sr-poor, mixed Kea–Loihi component. Given the lack of a hiatus between postshield and rejuvenated stages on Kauai, the rejuvenated mantle source is already able to melt at the tail end of shield stage activity and no secondary melting mechanism is required to explain the rejuvenated stage.

Description: Glycogen branching enzyme 1 (GBE1) plays an essential role in glycogen biosynthesis by generating α-1,6-glucosidic branches from α-1,4-linked glucose chains, to increase solubility of the glycogen polymer. Mutations in the GBE1 gene lead to the heterogeneous early-onset glycogen storage disorder type IV (GSDIV) or the late-onset adult polyglucosan body disease (APBD). To better understand this essential enzyme, we crystallized human GBE1 in the apo form, and in complex with a tetra- or hepta-saccharide. The GBE1 structure reveals a conserved amylase core that houses the active centre for the branching reaction and harbours almost all GSDIV and APBD mutations. A non-catalytic binding cleft, proximal to the site of the common APBD mutation p.Y329S, was found to bind the tetra- and hepta-saccharides and may represent a higher-affinity site employed to anchor the complex glycogen substrate for the branching reaction. Expression of recombinant GBE1-p.Y329S resulted in drastically reduced protein yield and solubility compared with wild type, suggesting this disease allele causes protein misfolding and may be amenable to small molecule stabilization. To explore this, we generated a structural model of GBE1-p.Y329S and designed peptides ab initio to stabilize the mutation. As proof-of-principle, we evaluated treatment of one tetra-peptide, Leu-Thr-Lys-Glu, in APBD patient cells. We demonstrate intracellular transport of this peptide, its binding and stabilization of GBE1-p.Y329S, and 2-fold increased mutant enzymatic activity compared with untreated patient cells. Together, our data provide the rationale and starting point for the screening of small molecule chaperones, which could become novel therapies for this disease.

Description: Essential tremor (ET) is a common movement disorder with an estimated prevalence of 5% of the population aged over 65 years. In spite of intensive efforts, the genetic architecture of ET remains unknown. We used a combination of whole-exome sequencing and targeted resequencing in three ET families. In vitro and in vivo experiments in oligodendrocyte precursor cells and zebrafish were performed to test our findings. Whole-exome sequencing revealed a missense mutation in TENM4 segregating in an autosomal-dominant fashion in an ET family. Subsequent targeted resequencing of TENM4 led to the discovery of two novel missense mutations. Not only did these two mutations segregate with ET in two additional families, but we also observed significant over transmission of pathogenic TENM4 alleles across the three families. Consistent with a dominant mode of inheritance, in vitro analysis in oligodendrocyte precursor cells showed that mutant proteins mislocalize. Finally, expression of human mRNA harboring any of three patient mutations in zebrafish embryos induced defects in axon guidance, confirming a dominant-negative mode of action for these mutations. Our genetic and functional data, which is corroborated by the existence of a Tenm4 knockout mouse displaying an ET phenotype, implicates TENM4 in ET. Together with previous studies of TENM4 in model organisms, our studies intimate that processes regulating myelination in the central nervous system and axon guidance might be significant contributors to the genetic burden of this disorder.

Description: Trisomy 21 causes skeletal alterations in individuals with Down syndrome (DS), but the causative trisomic gene and a therapeutic approach to rescue these abnormalities are unknown. Individuals with DS display skeletal alterations including reduced bone mineral density, modified bone structure and distinctive facial features. Due to peripheral skeletal anomalies and extended longevity, individuals with DS are increasingly more susceptible to bone fractures. Understanding the genetic and developmental origin of DS skeletal abnormalities would facilitate the development of therapies to rescue these and other deficiencies associated with DS. DYRK1A is found in three copies in individuals with DS and Ts65Dn DS mice and has been hypothesized to be involved in many Trisomy 21 phenotypes including skeletal abnormalities. Return of Dyrk1a copy number to normal levels in Ts65Dn mice rescued the appendicular bone abnormalities, suggesting that appropriate levels of DYRK1A expression are critical for the development and maintenance of the DS appendicular skeleton. Therapy using the DYRK1A inhibitor epigallocatechin-3-gallate improved Ts65Dn skeletal phenotypes. These outcomes suggest that the osteopenic phenotype associated with DS may be rescued postnatally by targeting trisomic Dyrk1a .

Description: Understanding the mechanisms of chromosomal double-strand break repair (DSBR) provides insight into genome instability, oncogenesis and genome engineering, including disease gene correction. Research into DSBR exploits rare-cutting endonucleases to cleave exogenous reporter constructs integrated into the genome. Multiple reporter constructs have been developed to detect various DSBR pathways. Here, using a single endogenous reporter gene, the X-chromosomal disease gene encoding hypoxanthine phosphoribosyltransferase ( HPRT ), we monitor the relative utilization of three DSBR pathways following cleavage by I-Sce I or CRISPR/Cas9 nucleases. For I-Sce I, our estimated frequencies of accurate or mutagenic non-homologous end-joining and gene correction by homologous recombination are 4.1, 1.5 and 0.16%, respectively. Unexpectedly, I-Sce I and Cas9 induced markedly different DSBR profiles. Also, using an I-Sce I-sensitive HPRT minigene, we show that gene correction is more efficient when using long double-stranded DNA than single- or double-stranded oligonucleotides. Finally, using both endogenous HPRT and exogenous reporters, we validate novel cell cycle phase-specific I-Sce I derivatives for investigating cell cycle variations in DSBR. The results obtained using these novel approaches provide new insights into template design for gene correction and the relationships between multiple DSBR pathways at a single endogenous disease gene.

Description: Despite recent progress in the characterization of genetic loci associated with multiple sclerosis (MS) risk, the ubiquitous linkage disequilibrium operating across the genome has stalled efforts to distinguish causative variants from proxy single-nucleotide polymorphisms (SNPs). Here, we have identified through fine mapping and meta-analysis EVI5 as the most plausible disease risk gene within the 1p22.1 locus. We further show that an exonic SNP associated with risk induces changes in superficial hydrophobicity patterns of the coiled-coil domain of EVI5, which, in turns, affects the EVI5 interactome. Immunoprecipitation of wild-type and mutated EVI5 followed by mass spectrometry generated a roster of disease-specific interactors functionally linked to lipid metabolism. Among the exclusive binding partners of the risk variant, we describe the novel interaction with sphingosine 1-phosphate lyase (SGPL1)—a key enzyme for the creation of the sphingosine-1 phosphate gradient, which is relevant to the pathogenic process and therapeutic management of MS.

Description: Hereditary sensory and autonomic neuropathy type 1 (HSAN1) is characterized by a loss of distal peripheral sensory and motorneuronal function, neuropathic pain and tissue necrosis. The most common cause of HSAN1 is due to dominant mutations in serine palmitoyl-transferase subunit 1 (SPT1). SPT catalyses the condensation of serine with palmitoyl-CoA, the initial step in sphingolipid biogenesis. Identified mutations in SPT1 are known to both reduce sphingolipid synthesis and generate catalytic promiscuity, incorporating alanine or glycine into the precursor sphingolipid to generate a deoxysphingoid base (DSB). Why either loss of function in SPT1 , or generation of DSBs should generate deficits in distal sensory function remains unclear. To address these questions, we generated a Drosophila model of HSAN1. Expression of dSpt1 bearing a disease-related mutation induced morphological deficits in synapse growth at the larval neuromuscular junction consistent with a dominant-negative action. Expression of mutant dSpt1 globally was found to be mildly toxic, but was completely toxic when the diet was supplemented with alanine, when DSBs were observed in abundance. Expression of mutant dSpt1 in sensory neurons generated developmental deficits in dendritic arborization with concomitant sensory deficits. A membrane trafficking defect was observed in soma of sensory neurons expressing mutant dSpt1 , consistent with endoplasmic reticulum (ER) to Golgi block. We found that we could rescue sensory function in neurons expressing mutant dSpt1 by co-expressing an effector of ER–Golgi function, Rab1 suggesting compromised ER function in HSAN1 affected dendritic neurons. Our Drosophila model identifies a novel strategy to explore the pathological mechanisms of HSAN1.

Description: Defective lysosomal acid β-glucosidase (GCase) in Gaucher disease causes accumulation of glucosylceramide (GC) and glucosylsphingosine (GS) that distress cellular functions. To study novel pathological mechanisms in neuronopathic Gaucher disease (nGD), a mouse model (4L;C*), an analogue to subacute human nGD, was investigated for global profiles of differentially expressed brain mRNAs (DEGs) and miRNAs (DEmiRs). 4L;C* mice displayed accumulation of GC and GS, activated microglial cells, reduced number of neurons and aberrant mitochondrial function in the brain followed by deterioration in motor function. DEGs and DEmiRs were characterized from sequencing of mRNA and miRNA from cerebral cortex, brain stem, midbrain and cerebellum of 4L;C* mice. Gene ontology enrichment and pathway analysis showed preferential mitochondrial dysfunction in midbrain and uniform inflammatory response and identified novel pathways, axonal guidance signaling, synaptic transmission, eIF2 and mammalian target of rapamycin (mTOR) signaling potentially involved in nGD. Similar analyses were performed with mice treated with isofagomine (IFG), a pharmacologic chaperone for GCase. IFG treatment did not alter the GS and GC accumulation significantly but attenuated the progression of the disease and altered numerous DEmiRs and target DEGs to their respective normal levels in inflammation, mitochondrial function and axonal guidance pathways, suggesting its regulation on miRNA and the associated mRNA that underlie the neurodegeneration in nGD. These analyses demonstrate that the neurodegenerative phenotype in 4L;C* mice was associated with dysregulation of brain mRNAs and miRNAs in axonal guidance, synaptic plasticity, mitochondria function, eIF2 and mTOR signaling and inflammation and provides new insights for the nGD pathological mechanism.

Description: Fragile X-associated disorders are Repeat Expansion Diseases that result from expansion of a CGG/CCG-repeat in the FMR1 gene. Contractions of the repeat tract also occur, albeit at lower frequency. However, these contractions can potentially modulate disease symptoms or generate an allele with repeat numbers in the normal range. Little is known about the expansion mechanism and even less about contractions. We have previously demonstrated that the mismatch repair (MMR) protein MSH2 is required for expansions in a mouse model of these disorders. Here, we show that MSH3, the MSH2-binding partner in the MutSβ complex, is required for 98% of germ line expansions and all somatic expansions in this model. In addition, we provide evidence for two different contraction mechanisms that operate in the mouse model, a MutSβ-independent one that generates small contractions and a MutSβ-dependent one that generates larger ones. We also show that MutSβ complexes formed with the repeats have altered kinetics of ATP hydrolysis relative to complexes with bona fide MMR substrates and that MutSβ increases the stability of the CCG-hairpins at physiological temperatures. These data may have important implications for our understanding of the mechanism(s) of repeat instability and for the role of MMR proteins in this process.

Description: Amyloid-β (Aβ) peptides originating from β-amyloid precursor protein (APP) are critical in Alzheimer's disease (AD). Cellular cholesterol levels/distribution can regulate production and clearance of Aβ peptides, albeit with contradictory outcomes. To better understand the relationship between cholesterol homeostasis and APP/Aβ metabolism, we have recently generated a bigenic ANPC mouse line overexpressing mutant human APP in the absence of Niemann-Pick type C-1 protein required for intracellular cholesterol transport. Using this unique bigenic ANPC mice and complementary stable N2a cells, we have examined the functional consequences of cellular cholesterol sequestration in the endosomal–lysosomal system, a major site of Aβ production, on APP/Aβ metabolism and its relation to neuronal viability. Levels of APP C-terminal fragments (α-CTF/β-CTF) and Aβ peptides, but not APP mRNA/protein or soluble APPα/APPβ, were increased in ANPC mouse brains and N2a-ANPC cells. These changes were accompanied by reduced clearance of peptides and an increased level/activity of -secretase, suggesting that accumulation of APP-CTFs is due to decreased turnover, whereas increased Aβ levels may result from a combination of increased production and decreased turnover. APP-CTFs and Aβ peptides were localized primarily in early-/late-endosomes and to some extent in lysosomes/autophagosomes. Cholesterol sequestration impaired endocytic-autophagic-lysosomal, but not proteasomal, clearance of APP-CTFs/Aβ peptides. Moreover, markers of oxidative stress were increased in vulnerable brain regions of ANPC mice and enhanced β-CTF/Aβ levels increased susceptibility of N2a-ANPC cells to H 2 O 2 -induced toxicity. Collectively, our results show that cellular cholesterol sequestration plays a key role in APP/Aβ metabolism and increasing neuronal vulnerability to oxidative stress in AD-related pathology.

Description: Alternative polyadenylation (APA) plays a role in gene expression regulation generally by shortening of 3'UTRs (untranslated regions) upon proliferative signals and relieving microRNA-mediated repression. Owing to high proliferative indices of triple negative breast cancers (TNBCs), we hypothesized APA to cause 3'UTR length changes in this aggressive subgroup of breast cancers. Our probe-based meta-analysis approach identified 3'UTR length alterations where the significant majority was shortening events (~70%, 113 of 165) of mostly proliferation-related transcripts in 520 TNBC patients compared with controls. Representative shortening events were further investigated for their microRNA binding potentials by computational predictions and dual-luciferase assay. In silico -predicted 3'UTR shortening events were experimentally confirmed in patient and cell line samples. To begin addressing the underlying mechanisms, we found CSTF2 (cleavage stimulation factor 2), a major regulator of 3'UTR shortening to be up-regulated in response to epidermal growth factor (EGF). EGF treatment also resulted with further shortening of the 3'UTRs. To investigate the contribution of CSTF2 and 3'UTR length alterations to the proliferative phenotype, we showed pharmacological inhibition of the EGF pathway to lead to a reduction in CSTF2 levels. Accordingly, RNAi-induced silencing of CSTF2 decreased the proliferative rate of cancer cells. Therefore, our computational and experimental approach revealed a pattern of 3'UTR length changes in TNBC patients and a potential link between APA and EGF signaling. Overall, detection of 3'UTR length alterations of various genes may help the discovery of new cancer-related genes, which may have been overlooked in conventional microarray gene expression analyses.

Description: Usher syndrome (USH) is the leading cause of inherited deaf-blindness, with type 2 (USH2) being the most common clinical form. Studies suggest that proteins encoded by USH2 causative genes assemble into the ankle link complex (ALC) at the hair cell stereociliary bundle; however, little is known about the in vivo assembly and function of this complex. Using various USH2 mutant mice, we showed by immunofluorescence that USH2 proteins play different roles in cochlear ALC assembly, with G protein-coupled receptor 98 being the most important protein. Complex assembly likely occurs at the stereociliary bundle but not along the protein transport route in the cell body. Stereociliary morphological defects in USH2 mutant mice suggest roles for the ALC in regulating inner hair cell stereociliary growth and differentiation as well as outer hair cell stereociliary rigidity and organization during development. These roles are unique from the bundle cohesion role of Usher syndrome type 1 protein complexes. Loss of individual USH2 gene expressions leads to variable morphological and functional consequences, correlating with the severity of ALC disruption. This finding suggests a potential genotype–phenotype correlation in USH2 patients. In summary, this study provides novel insights into the molecular mechanism underlying cochlear stereociliary bundle development and hearing loss pathogenesis of various USH2 subtypes. Our thorough phenotypical characterization of USH2 mouse models is essential for future use of these animal models in therapeutic development.

Description: Assisted reproductive technologies (ART) are associated with several complications including low birth weight, abnormal placentation and increased risk for rare imprinting disorders. Indeed, experimental studies demonstrate ART procedures independent of existing infertility induce epigenetic perturbations in the embryo and extraembryonic tissues. To test the hypothesis that these epigenetic perturbations persist and result in adverse outcomes at term, we assessed placental morphology and methylation profiles in E18.5 mouse concepti generated by in vitro fertilization (IVF) in two different genetic backgrounds. We also examined embryo transfer (ET) and superovulation procedures to ascertain if they contribute to developmental and epigenetic effects. Increased placental weight and reduced fetal-to-placental weight ratio were observed in all ART groups when compared with naturally conceived controls, demonstrating that non-surgical embryo transfer alone can impact placental development. Furthermore, superovulation further induced overgrowth of the placental junctional zone. Embryo transfer and superovulation defects were limited to these morphological changes, as we did not observe any differences in epigenetic profiles. IVF placentae, however, displayed hypomethylation of imprinting control regions of select imprinted genes and a global reduction in DNA methylation levels. Although we did not detect significant differences in DNA methylation in fetal brain or liver samples, rare IVF concepti displayed very low methylation and abnormal gene expression from the normally repressed allele. Our findings suggest that individual ART procedures cumulatively increase placental morphological abnormalities and epigenetic perturbations, potentially causing adverse neonatal and long-term health outcomes in offspring.

Description: Mutations in subunits or regulators of cohesin cause a spectrum of disorders in humans known as the ‘cohesinopathies’. Cohesinopathies, including the best known example Cornelia de Lange syndrome (CdLS), are characterized by broad spectrum, multifactorial developmental anomalies. Heart defects occur at high frequency and can reach up to 30% in CdLS. The mechanisms by which heart defects occur are enigmatic, but assumed to be developmental in origin. In this study, we depleted cohesin subunit Rad21 by 70–80% in a zebrafish cohesinopathy model. The hearts of Rad21-depleted animals were smaller, often failed to loop, and functioned less efficiently than size-matched controls. Functional deficiency was accompanied by valve defects and reduced ejection fraction. Interestingly, neural crest cells failed to populate the heart and instead exhibited a wandering behavior. Consequently, these cells also failed to condense correctly into pharyngeal arches. Transcriptome analysis revealed that Wnt pathway, chemokine and cadherin genes are dysregulated at the time of cardiac neural crest development. Our results give insight into the etiology of heart defects in the cohesinopathies, and raise the possibility that mild mutations in cohesin genes may be causative of a fraction of congenital heart disease in human populations.

Description: The DFNB31 gene plays an indispensable role in the cochlea and retina. Mutations in this gene disrupt its various isoforms and lead to non-syndromic deafness, blindness and deaf-blindness. However, the known expression of Dfnb31 , the mouse ortholog of DFNB31 , in vestibular organs and the potential vestibular-deficient phenotype observed in one Dfnb31 mutant mouse ( Dfnb31 wi/wi ) suggest that DFNB31 may also be important for vestibular function. In this study, we find that full-length (FL-) and C-terminal (C-) whirlin isoforms are expressed in the vestibular organs, where their stereociliary localizations are similar to those of developing cochlear inner hair cells. No whirlin is detected in Dfnb31 wi/wi vestibular organs, while only C-whirlin is expressed in Dfnb31 neo/neo vestibular organs. Both FL- and C-whirlin isoforms are required for normal vestibular stereociliary growth, although they may play slightly different roles in the central and peripheral zones of the crista ampullaris. Vestibular sensory-evoked potentials demonstrate severe to profound vestibular deficits in Dfnb31 neo/neo and Dfnb31 wi/wi mice. Swimming and rotarod tests demonstrate that the two Dfnb31 mutants have balance problems, with Dfnb31 wi/wi mice being more affected than Dfnb31 neo/neo mice. Because Dfnb31 wi/wi and Dfnb31 neo/neo mice faithfully recapitulate hearing and vision symptoms in patients, our findings of vestibular dysfunction in these Dfnb31 mutants raise the question of whether DFNB31 -deficient patients may acquire vestibular as well as hearing and vision loss.

Description: Methylmalonic acidurias (MMAurias) are a group of inherited disorders in the catabolism of branched-chain amino acids, odd-chain fatty acids and cholesterol caused by complete or partial deficiency of methylmalonyl-CoA mutase ( mut 0 and mut - subtype respectively) and by defects in the metabolism of its cofactor 5'-deoxyadenosylcobalamin ( cblA , cblB or cblD variant 2 type). A long-term complication found in patients with mut 0 and cblB variant is chronic tubulointerstitial nephritis. The underlying pathomechanism has remained unknown. We established an in vitro model of tubular epithelial cells from patient urine (hTEC; 9 controls, 5 mut 0 , 1 cblB ). In all human tubular epithelial cell (hTEC) lines we found specific tubular markers (AQP1, UMOD, AQP2). Patient cells showed disturbance of energy metabolism in glycolysis, mitochondrial respiratory chain and Krebs cycle in concert with increased reactive oxygen species (ROS) formation. Electron micrographs indicated increased autophagosome production and endoplasmic reticulum stress, which was supported by positive acridine orange staining and elevated levels of LC3 II, P62 and pIRE1. Screening mTOR signaling revealed a release of inhibition of autophagy. Patient hTEC produced and secreted elevated amounts of the pro-inflammatory cytokine IL8, which was highly correlated with the acridine orange staining. Summarizing, hTEC of MMAuria patients are characterized by disturbed energy metabolism and ROS production that lead to increased autophagy and IL8 secretion.

Description: RNA dysregulation is a newly recognized disease mechanism in amyotrophic lateral sclerosis (ALS). Here we identify Drosophila fragile X mental retardation protein (dFMRP) as a robust genetic modifier of TDP-43-dependent toxicity in a Drosophila model of ALS. We find that dFMRP overexpression (dFMRP OE) mitigates TDP-43 dependent locomotor defects and reduced lifespan in Drosophila. TDP-43 and FMRP form a complex in flies and human cells. In motor neurons, TDP-43 expression increases the association of dFMRP with stress granules and colocalizes with polyA binding protein in a variant-dependent manner. Furthermore, dFMRP dosage modulates TDP-43 solubility and molecular mobility with overexpression of dFMRP resulting in a significant reduction of TDP-43 in the aggregate fraction. Polysome fractionation experiments indicate that dFMRP OE also relieves the translation inhibition of futsch mRNA, a TDP-43 target mRNA, which regulates neuromuscular synapse architecture. Restoration of futsch translation by dFMRP OE mitigates Futsch-dependent morphological phenotypes at the neuromuscular junction including synaptic size and presence of satellite boutons. Our data suggest a model whereby dFMRP is neuroprotective by remodeling TDP-43 containing RNA granules, reducing aggregation and restoring the translation of specific mRNAs in motor neurons.

Description: Friedreich's ataxia (FRDA) is a severe neurodegenerative disease caused by homozygous expansion of the guanine-adenine-adenine (GAA) repeats in intron 1 of the FXN gene leading to transcriptional repression of frataxin expression. Post-translational histone modifications that typify heterochromatin are enriched in the vicinity of the repeats, whereas active chromatin marks in this region are underrepresented in FRDA samples. Yet, the immediate effect of the expanded repeats on transcription progression through FXN and their long-range effect on the surrounding genomic context are two critical questions that remain unanswered in the molecular pathogenesis of FRDA. To address these questions, we conducted next-generation RNA sequencing of a large cohort of FRDA and control primary fibroblasts. This comprehensive analysis revealed that the GAA-induced silencing effect does not influence expression of neighboring genes upstream or downstream of FXN . Furthermore, no long-range silencing effects were detected across a large portion of chromosome 9. Additionally, results of chromatin immunoprecipitation studies confirmed that histone modifications associated with repressed transcription are confined to the FXN locus. Finally, deep sequencing of FXN pre-mRNA molecules revealed a pronounced defect in the transcription elongation rate in FRDA cells when compared with controls. These results indicate that approaches aimed to reactivate frataxin expression should simultaneously address deficits in transcription initiation and elongation at the FXN locus.

Description: Retinal degeneration and visual impairment are the first signs of juvenile neuronal ceroid lipofuscinosis caused by CLN3 mutations, followed by inevitable progression to blindness. We investigated retinal degeneration in Cln3 ex1-6 null mice, revealing classic ‘fingerprint’ lysosomal storage in the retinal pigment epithelium (RPE), replicating the human disease. The lysosomes contain mitochondrial F 0 -ATP synthase subunit c along with undigested membranes, indicating a reduced degradative capacity. Mature autophagosomes and basal phagolysosomes, the terminal degradative compartments of autophagy and phagocytosis, are also increased in Cln3 ex1 - 6 RPE, reflecting disruption to these key pathways that underpin the daily phagocytic turnover of photoreceptor outer segments (POS) required for maintenance of vision. The accumulated autophagosomes have post-lysosome fusion morphology, with undigested internal contents visible, while accumulated phagosomes are frequently docked to cathepsin D-positive lysosomes, without mixing of phagosomal and lysosomal contents. This suggests lysosome-processing defects affect both autophagy and phagocytosis, supported by evidence that phagosomes induced in Cln3 ex1 - 6 -derived mouse embryonic fibroblasts have visibly disorganized membranes, unprocessed internal vesicles and membrane contents, in addition to reduced LAMP1 membrane recruitment. We propose that defective lysosomes in Cln3 ex1 - 6 RPE have a reduced degradative capacity that impairs the final steps of the intimately connected autophagic and phagocytic pathways that are responsible for degradation of POS. A build-up of degradative organellar by-products and decreased recycling of cellular materials is likely to disrupt processes vital to maintenance of vision by the RPE.

Description: Human gene mutations have revealed that a significant number of ADAMTS (a disintegrin-like and metalloproteinase (reprolysin type) with thrombospondin type 1 motifs) proteins are necessary for normal ocular development and eye function. Mutations in human ADAMTSL4 , encoding an ADAMTS-like protein which has been implicated in fibrillin microfibril biogenesis, cause ectopia lentis (EL) and EL et pupillae. Here, we report the first ADAMTSL4 mouse model, tvrm267 , bearing a nonsense mutation in Adamtsl4 . Homozygous Adamtsl4 tvrm267 mice recapitulate the EL phenotype observed in humans, and our analysis strongly suggests that ADAMTSL4 is required for stable anchorage of zonule fibers to the lens capsule. Unexpectedly, homozygous Adamtsl4 tvrm267 mice exhibit focal retinal pigment epithelium (RPE) defects primarily in the inferior eye. RPE dedifferentiation was indicated by reduced pigmentation, altered cellular morphology and a reduction in RPE-specific transcripts. Finally, as with a subset of patients with ADAMTSL4 mutations, increased axial length, relative to age-matched controls, was observed and was associated with the severity of the RPE phenotype. In summary, the Adamtsl4 tvrm267 model provides a valuable tool to further elucidate the molecular basis of zonule formation, the pathophysiology of EL and ADAMTSL4 function in the maintenance of the RPE.

Description: Leber's hereditary optic neuropathy (LHON) is a maternally inherited blinding disease characterized by degeneration of retinal ganglion cells (RGCs) and consequent optic nerve atrophy. Peculiar features of LHON are incomplete penetrance and gender bias, with a marked male prevalence. Based on the different hormonal metabolism between genders, we proposed that estrogens play a protective role in females and showed that these hormones ameliorate mitochondrial dysfunction in LHON through the estrogen receptors (ERs). We also showed that ERβ localize to the mitochondria of RGCs. Thus, targeting ERβ may become a therapeutic strategy for LHON specifically aimed at avoiding or delaying the onset of disease in mutation carriers. Here, we tested the effects of ERβ targeting on LHON mitochondrial defective metabolism by treating LHON cybrid cells carrying the m.11778G〉A mutation with a combination of natural estrogen-like compounds that bind ERβ with high selectivity. We demonstrated that these molecules improve cell viability by reducing apoptosis, inducing mitochondrial biogenesis and strongly reducing the levels of reactive oxygen species in LHON cells. These effects were abolished in cells with ERβ knockdown by silencing receptor expression or by using specific receptor antagonists. Our observations support the hypothesis that estrogen-like molecules may be useful in LHON prophylactic therapy. This is particularly important for lifelong disease prevention in unaffected LHON mutation carriers. Current strategies attempting to combat degeneration of RGCs during the acute phase of LHON have not been very effective. Implementing a different and preemptive approach with a low risk profile may be very helpful.

Description: Mucopolysaccharidosis-I (MPS-I) is a lysosomal storage disease (LSD) caused by inactivating mutations of IDUA , encoding the glycosaminoglycan-degrading enzyme α-l-iduronidase. Although MPS-I is associated with skeletal abnormalities, the impact of IDUA deficiency on bone remodeling is poorly defined. Here we report that Idua -deficient mice progressively develop a high bone mass phenotype with pathological lysosomal storage in cells of the osteoblast lineage. Histomorphometric quantification identified shortening of bone-forming units and reduced osteoclast numbers per bone surface. This phenotype was not transferable into wild-type mice by bone marrow transplantation (BMT). In contrast, the high bone mass phenotype of Idua -deficient mice was prevented by BMT from wild-type donors. At the cellular level, BMT did not only normalize defects of Idua -deficient osteoblasts and osteocytes but additionally caused increased osteoclastogenesis. Based on clinical observations in an individual with MPS-I, previously subjected to BMT and enzyme replacement therapy (ERT), we treated Idua -deficient mice accordingly and found that combining both treatments normalized all histomorphometric parameters of bone remodeling. Our results demonstrate that BMT and ERT profoundly affect skeletal remodeling of Idua -deficient mice, thereby suggesting that individuals with MPS-I should be monitored for their bone remodeling status, before and after treatment, to avoid long-term skeletal complications.

Description: The autism spectrum disorders (ASD) comprise a broad group of behaviorally related neurodevelopmental disorders affecting as many as 1 in 68 children. The hallmarks of ASD consist of impaired social and communication interactions, pronounced repetitive behaviors and restricted patterns of interests. Family, twin and epidemiological studies suggest a polygenetic and epistatic susceptibility model involving the interaction of many genes; however, the etiology of ASD is likely to be complex and include both epigenetic and environmental factors. 5-hydroxymethylcytosine (5hmC) is a novel environmentally sensitive DNA modification that is highly enriched in post-mitotic neurons and is associated with active transcription of neuronal genes. Here, we used an established chemical labeling and affinity purification method coupled with high-throughput sequencing technology to generate a genome-wide profile of striatal 5hmC in an autism mouse model ( Cntnap2 –/– mice) and found that at 9 weeks of age the Cntnap2 –/– mice have a genome-wide disruption in 5hmC, primarily in genic regions and repetitive elements. Annotation of differentially hydroxymethylated regions (DhMRs) to genes revealed a significant overlap with known ASD genes (e.g. Nrxn1 and Reln ) that carried an enrichment of neuronal ontological functions, including axonogenesis and neuron projection morphogenesis. Finally, sequence motif predictions identified associations with transcription factors that have a high correlation with important genes in neuronal developmental and functional pathways. Together, our data implicate a role for 5hmC-mediated epigenetic modulation in the pathogenesis of autism and represent a critical step toward understanding the genome-wide molecular consequence of the Cntnap2 mutation, which results in an autism-like phenotype.

Description: Bladder exstrophy, a severe congenital urological malformation when a child is born with an open urinary bladder, is the most common form of bladder exstrophy-epispadias complex (BEEC) with an incidence of 1:30,000 children of Caucasian descent. Recent studies suggest that WNT genes may contribute to the etiology of bladder exstrophy. Here, we evaluated WNT -pathway genes in 20 bladder exstrophy patients using massively parallel sequencing. In total 13 variants were identified in WNT3 , WNT6 , WNT7A , WNT8B , WNT10A , WNT11 , WNT16 , FZD5 , LRP1 and LRP10 genes and predicted as potentially disease causing, of which seven variants were novel. One variant, identified in a patient with a de novo nonsynonymous substitution in WNT3 (p.Cys91Arg), was further evaluated in zebrafish. Knock down of wnt3 in zebrafish showed cloaca malformations, including disorganization of the cloaca epithelium and expansion of the cloaca lumen. Our study suggests that the function of the WNT3 p.Cys91Arg variant was altered, since RNA overexpression of mutant Wnt3 RNA does not result in embryonic lethality as seen with wild-type WNT3 mRNA. Finally, we also mutation screened the WNT3 gene further in 410 DNA samples from BEEC cases and identified one additional mutation c.638G〉A (p.Gly213Asp), which was paternally inherited. In aggregate our data support the involvement of WNT -pathway genes in BEEC and suggest that WNT3 in itself is a rare cause of BEEC.

Description: Multiple symmetric lipomatosis (MSL) is a mitochondrial disorder with impaired brown fat metabolism that has been associated with MERRF mutations in some, but not all, patients. We studied a sibling pair and an unrelated indiviadual who presented with MSL and neuropathy to determine the genetic etiology of this disorder in patients who did not carry the MSL-associated MERRF mutation. Whole-exome sequencing was performed on the siblings, and a rare, shared homozygous mutation in MFN2 (c.2119C〉T: p.R707W) was identified. The mutation was not present in their healthy siblings. In silico programs predict it to be pathogenic, and heterozygous carriers of the MFN2 p.R707W substitution are known to have Charcot–Marie–Tooth (CMT) disease. A third, unrelated patient with multiple symmetrical lipomatosis and neuropathy also harbored the same homozygous mutation and had been previously diagnosed with CMT. Functional studies in patient fibroblasts demonstrate that the p.R707W substitution impairs homotypic (MFN2–MFN2) protein interactions required for normal activity and renders mitochondria prone to perinuclear aggregation. These findings show that homozygous mutations at p.R707W in MFN2 are a novel cause of multiple symmetrical lipomatosis.

Description: The adaptor protein-2 sigma subunit (AP22) is pivotal for clathrin-mediated endocytosis of plasma membrane constituents such as the calcium-sensing receptor (CaSR). Mutations of the AP22 Arg15 residue result in familial hypocalciuric hypercalcaemia type 3 (FHH3), a disorder of extracellular calcium (Ca 2+ o ) homeostasis. To elucidate the role of AP22 in Ca 2+ o regulation, we investigated 65 FHH probands, without other FHH-associated mutations, for AP22 mutations, characterized their functional consequences and investigated the genetic mechanisms leading to FHH3. AP22 mutations were identified in 17 probands, comprising 5 Arg15Cys, 4 Arg15His and 8 Arg15Leu mutations. A genotype–phenotype correlation was observed with the Arg15Leu mutation leading to marked hypercalcaemia. FHH3 probands harboured additional phenotypes such as cognitive dysfunction. All three FHH3-causing AP22 mutations impaired CaSR signal transduction in a dominant-negative manner. Mutational bias was observed at the AP22 Arg15 residue as other predicted missense substitutions (Arg15Gly, Arg15Pro and Arg15Ser), which also caused CaSR loss-of-function, were not detected in FHH probands, and these mutations were found to reduce the numbers of CaSR-expressing cells. FHH3 probands had significantly greater serum calcium (sCa) and magnesium (sMg) concentrations with reduced urinary calcium to creatinine clearance ratios (CCCR) in comparison with FHH1 probands with CaSR mutations, and a calculated index of sCa x sMg/100 x CCCR, which was ≥ 5.0, had a diagnostic sensitivity and specificity of 83 and 86%, respectively, for FHH3. Thus, our studies demonstrate AP22 mutations to result in a more severe FHH phenotype with genotype–phenotype correlations, and a dominant-negative mechanism of action with mutational bias at the Arg15 residue.

Description: Sprouty proteins are regulators of cell growth and branching morphogenesis. Unlike mouse Spry3 , which is X-linked, human SPRY3 maps to the pseudoautosomal region 2; however, the human Y-linked allele is not expressed due to epigenetic silencing by an unknown mechanism. SPRY3 maps adjacent to X-linked Trimethyllysine hydroxylase epsilon ( TMLHE ), recently identified as an autism susceptibility gene. We report that Spry3 is highly expressed in central and peripheral nervous system ganglion cells in mouse and human, including cerebellar Purkinje cells and retinal ganglion cells. Transient over-expression or knockdown of Spry3 in cultured mouse superior cervical ganglion cells inhibits and promotes, respectively, neurite growth and branching. A 0.7 kb gene fragment spanning the human SPRY3 transcriptional start site recapitulates the endogenous Spry3 -expression pattern in LacZ reporter mice. In the human and mouse the SPRY3 promoter contains an AG-rich repeat and we found co-expression, and promoter binding and/or regulation of SPRY3 expression by transcription factors MAZ, EGR1, ZNF263 and PAX6. We identified eight alleles of the human SPRY3 promoter repeat in Caucasians, and similar allele frequencies in autism families. We characterized multiple SPRY3 transcripts originating at two CpG islands in the X-linked F8A3 — TMLHE region, suggesting X chromosome regulation of SPRY3 . These findings provide an explanation for differential regulation of X and Y-linked SPRY3 alleles. In addition, the presence of a SPRY3 transcript exon in a previously described X chromosome deletion associated with autism, and the cerebellar interlobular variation in Spry3 expression coincident with the reported pattern of Purkinje cell loss in autism, suggest SPRY3 as a candidate susceptibility locus for autism.

Description: Glycogen storage disease type-Ia (GSD-Ia) is caused by a lack of glucose-6-phosphatase-α (G6Pase-α or G6PC) activity. We have shown that gene therapy mediated by a recombinant adeno-associated virus (rAAV) vector expressing human G6Pase-α normalizes blood glucose homeostasis in the global G6pc knockout ( G6pc –/– ) mice for 70–90 weeks. The treated G6pc –/– mice expressing 3–63% of normal hepatic G6Pase-α activity (AAV mice) produce endogenous hepatic glucose levels 61–68% of wild-type littermates, have a leaner phenotype and exhibit fasting blood insulin levels more typical of young adult mice. We now show that unlike wild-type mice, the lean AAV mice have increased caloric intake and do not develop age-related obesity or insulin resistance. Pathway analysis shows that signaling by hepatic carbohydrate response element binding protein that improves glucose tolerance and insulin signaling is activated in AAV mice. In addition, several longevity factors in the calorie restriction pathway, including the NADH shuttle systems, NAD + concentrations and the AMP-activated protein kinase/sirtuin 1/peroxisome proliferator-activated receptor- coactivator 1α pathway are upregulated in the livers of AAV mice. The finding that partial restoration of hepatic G6Pase-α activity in GSD-Ia mice not only attenuates the phenotype of hepatic G6Pase-α deficiency but also prevents the development of age-related obesity and insulin resistance seen in wild-type mice may suggest relevance of the G6Pase-α enzyme to obesity and diabetes.

Description: Congenital Hyperinsulinism (CHI) is a rare heterogeneous disease characterized by unregulated insulin secretion. Dominant mutations in ABCC8 causing medically unresponsive CHI have been reported; however, the molecular mechanisms are not clear. The molecular basis of medically unresponsive CHI due to dominant ABCC8 mutations has been studied in 10 patients, who were medically unresponsive to diazoxide (DZX), and nine of whom required a near-total pancreatectomy, and one partial pancreatectomy. DNA sequencing revealed seven dominant inactivating heterozygous missense mutations in ABCC8 , including one novel and six previously reported but uncharacterized mutations. Two groups of mutations with different cellular mechanisms were characterized. Mutations in the transmembrane domain (TMD) were more responsive to channel activators such as DZX, MgADP and metabolic inhibition. The trafficking analysis has shown that nucleotide-binding domain two (NBD2) mutations are not retained in the endoplasmic reticulum (ER) and are present on the membrane. However, the TMD mutations were retained in the ER. D1506E was the most severe SUR1-NBD2 mutation. Homologous expression of D1506E revealed a near absence of K ATP currents in the presence of DZX and intracellular MgADP. Heterozygous expression of D1506E showed a strong dominant-negative effect on SUR1\K ir 6.2 currents. Overall, we define two groups of mutation with different cellular mechanisms. In the first group, channel complexes with mutations in NBD2 of SUR1 traffic normally but are unable to be activated by MgADP. In the second group, channels mutations in the TMD of SUR1 are retained in the ER and have variable functional impairment.

Description: Fanconi anemia (FA) is a rare inherited disorder clinically characterized by congenital malformations, progressive bone marrow failure and cancer susceptibility. At the cellular level, FA is associated with hypersensitivity to DNA-crosslinking genotoxins. Eight of 17 known FA genes assemble the FA E3 ligase complex, which catalyzes monoubiquitination of FANCD2 and is essential for replicative DNA crosslink repair. Here, we identify the first FA patient with biallelic germline mutations in the ubiquitin E2 conjugase UBE2T. Both mutations were aluY-mediated: a paternal deletion and maternal duplication of exons 2–6. These loss-of-function mutations in UBE2T induced a cellular phenotype similar to biallelic defects in early FA genes with the absence of FANCD2 monoubiquitination. The maternal duplication produced a mutant mRNA that could encode a functional protein but was degraded by nonsense-mediated mRNA decay. In the patient's hematopoietic stem cells, the maternal allele with the duplication of exons 2–6 spontaneously reverted to a wild-type allele by monoallelic recombination at the duplicated aluY repeat, thereby preventing bone marrow failure. Analysis of germline DNA of 814 normal individuals and 850 breast cancer patients for deletion or duplication of UBE2T exons 2–6 identified the deletion in only two controls, suggesting aluY-mediated recombinations within the UBE2T locus are rare and not associated with an increased breast cancer risk. Finally, a loss-of-function germline mutation in UBE2T was detected in a high-risk breast cancer patient with wild-type BRCA1/2 . Cumulatively, we identified UBE2T as a bona fide FA gene ( FANCT ) that also may be a rare cancer susceptibility gene.

Description: Myoclonus-dystonia (M-D) is a very rare movement disorder, caused in ~30–50% of cases by mutations in SGCE . The CACNA1B variant c.4166G〉A; (p.R1389H) was recently reported as the likely causative mutation in a single 3-generation Dutch pedigree with five subjects affected by a unique dominant M-D syndrome and cardiac arrhythmias. In an attempt to replicate this finding, we assessed by direct sequencing the frequency of CACNA1B c.4166G〉A; (p.R1389H) in a cohort of 520 M-D cases, in which SGCE mutations had been previously excluded. A total of 146 cases (28%) had a positive family history of M-D. The frequency of the variant was also assessed in 489 neurologically healthy controls and in publicly available data sets of genetic variation (1000 Genomes, Exome Variant Server and Exome Aggregation Consortium). The variant was detected in a single sporadic case with M-D, but in none of the 146 probands with familial M-D. Overall, the variant was present at comparable frequencies in M-D cases (1 out of 520; 0.19%) and healthy controls (1 out of 489; 0.2%). A similar frequency of the variant was also reported in all publicly available databases. These results do not support a causal association between the CACNA1B c.4166G〉A; (p.R1389H) variant and M-D.

Description: The alternative splicing of the tau gene, MAPT , generates six protein isoforms in the adult human central nervous system (CNS). Tau splicing is developmentally regulated and dysregulated in disease. Mutations in MAPT that alter tau splicing cause frontotemporal dementia (FTD) with tau pathology, providing evidence for a causal link between altered tau splicing and disease. The use of induced pluripotent stem cell (iPSC)-derived neurons has revolutionized the way we model neurological disease in vitro . However, as most tau mutations are located within or around the alternatively spliced exon 10, it is important that iPSC–neurons splice tau appropriately in order to be used as disease models. To address this issue, we analyzed the expression and splicing of tau in iPSC-derived cortical neurons from control patients and FTD patients with the 10 + 16 intronic mutation in MAPT . We show that control neurons only express the fetal tau isoform (0N3R), even at extended time points of 100 days in vitro . Neurons from FTD patients with the 10 + 16 mutation in MAPT express both 0N3R and 0N4R tau isoforms, demonstrating that this mutation overrides the developmental regulation of exon 10 inclusion in our in vitro model. Further, at extended time points of 365 days in vitro , we observe a switch in tau splicing to include six tau isoforms as seen in the adult human CNS. Our results demonstrate the importance of neuronal maturity for use in in vitro modeling and provide a system that will be important for understanding the functional consequences of altered tau splicing.

Description: Although DNA methylation is now recognized as an important mediator of complex diseases, the extent to which the genetic basis of such diseases is accounted for by DNA methylation is unknown. In the setting of large, extended families representing a minority, high-risk population of the USA, we aimed to characterize the role of epigenome-wide DNA methylation in type 2 diabetes (T2D). Using Illumina HumanMethylation450 BeadChip arrays, we tested for association of DNA methylation at 446 356 sites with age, sex and phenotypic traits related to T2D in 850 pedigreed Mexican-American individuals. Robust statistical analyses showed that (i) 15% of the methylome is significantly heritable, with a median heritability of 0.14; (ii) DNA methylation at 14% of CpG sites is associated with nearby sequence variants; (iii) 22% and 3% of the autosomal CpG sites are associated with age and sex, respectively; (iv) 53 CpG sites were significantly associated with liability to T2D, fasting blood glucose and insulin resistance; (v) DNA methylation levels at five CpG sites, mapping to three well-characterized genes ( TXNIP , ABCG1 and SAMD12 ) independently explained 7.8% of the heritability of T2D (vi) methylation at these five sites was unlikely to be influenced by neighboring DNA sequence variation. Our study has identified novel epigenetic indicators of T2D risk in Mexican Americans who have increased risk for this disease. These results provide new insights into potential treatment targets of T2D.

Description: Cumulative evidence indicates that the onset and severity of Huntington's disease (HD) symptoms correlate with connectivity deficits involving specific neuronal populations within cortical and basal ganglia circuits. Brain imaging studies and pathological reports further associated these deficits with alterations in cerebral white matter structure and axonal pathology. However, whether axonopathy represents an early pathogenic event or an epiphenomenon in HD remains unknown, nor is clear the identity of specific neuronal populations affected. To directly evaluate early axonal abnormalities in the context of HD in vivo , we bred transgenic YFP( J16 ) with R6/2 mice, a widely used HD model. Diffusion tensor imaging and fluorescence microscopy studies revealed a marked degeneration of callosal axons long before the onset of motor symptoms. Accordingly, a significant fraction of YFP-positive cortical neurons in YFP( J16 ) mice cortex were identified as callosal projection neurons. Callosal axon pathology progressively worsened with age and was influenced by polyglutamine tract length in mutant huntingtin (mhtt). Degenerating axons were dissociated from microscopically visible mhtt aggregates and did not result from loss of cortical neurons. Interestingly, other axonal populations were mildly or not affected, suggesting differential vulnerability to mhtt toxicity. Validating these results, increased vulnerability of callosal axons was documented in the brains of HD patients. Observations here provide a structural basis for the alterations in cerebral white matter structure widely reported in HD patients. Collectively, our data demonstrate a dying-back pattern of degeneration for cortical projection neurons affected in HD, suggesting that axons represent an early and potentially critical target for mhtt toxicity.

Description: Mutations in alpha- and beta-tubulins are increasingly recognized as a major cause of malformations of cortical development (MCD), typically lissencephaly, pachygyria and polymicrogyria; however, sequencing tubulin genes in large cohorts of MCD patients has detected tubulin mutations in only 1–13%. We identified patients with a highly characteristic cerebellar dysplasia but without lissencephaly, pachygyria and polymicrogyria typically associated with tubulin mutations. Remarkably, in seven of nine patients (78%), targeted sequencing revealed mutations in three different tubulin genes ( TUBA1A , TUBB2B  and TUBB3 ), occurring de novo or inherited from a mosaic parent. Careful re-review of the cortical phenotype on brain imaging revealed only an irregular pattern of gyri and sulci, for which we propose the term tubulinopathy-related dysgyria. Basal ganglia (100%) and brainstem dysplasia (80%) were common features. On the basis of in silico structural predictions, the mutations affect amino acids in diverse regions of the alpha-/beta-tubulin heterodimer, including the nucleotide binding pocket. Cell-based assays of tubulin dynamics reveal various effects of the mutations on incorporation into microtubules: TUBB3 p.Glu288Lys and p.Pro357Leu do not incorporate into microtubules at all, whereas TUBB2B p.Gly13Ala shows reduced incorporation and TUBA1A p.Arg214His incorporates fully, but at a slower rate than wild-type. The broad range of effects on microtubule incorporation is at odds with the highly stereotypical clinical phenotype, supporting differential roles for the three tubulin genes involved. Identifying this highly characteristic phenotype is important due to the low recurrence risk compared with the other (recessive) cerebellar dysplasias and the apparent lack of non-neurological medical issues.

Description: Preferential dysfunction/degeneration of midbrain substantia nigra pars compacta (SNpc) dopaminergic (DA) neurons contributes to the main movement symptoms manifested in Parkinson's disease (PD). Although the Leucine-rich repeat kinase 2 ( LRRK2 ) G2019S missense mutation ( LRRK2 G2019S) is the most common causative genetic factor linked to PD, the effects of LRRK2 G2019S on the function and survival of SNpc DA neurons are poorly understood. Using a binary gene expression system, we generated transgenic mice expressing either wild-type human LRRK2 (WT mice) or the LRRK2 G2019S mutation (G2019S mice) selectively in the midbrain DA neurons. Here we show that overexpression of LRRK2 G2019S did not induce overt motor abnormalities or substantial SNpc DA neuron loss. However, the LRRK2 G2019S mutation impaired dopamine homeostasis and release in aged mice. This reduction in dopamine content/release coincided with the degeneration of DA axon terminals and decreased expression of DA neuron-enriched genes tyrosine hydroxylase (TH), vesicular monoamine transporter 2, dopamine transporter and aldehyde dehydrogenase 1. These factors are responsible for dopamine synthesis, transport and degradation, and their expression is regulated by transcription factor paired-like homeodomain 3 (PITX3). Levels of Pitx3 mRNA and protein were similarly decreased in the SNpc DA neurons of aged G2019S mice. Together, these findings suggest that PITX3-dependent transcription regulation could be one of the many potential mechanisms by which LRRK2 G2019S acts in SNpc DA neurons, resulting in downregulation of its downstream target genes critical for dopamine homeostasis and release.

Description: Epidemiological studies have reported inconsistent associations between telomere length (TL) and risk for various cancers. These inconsistencies are likely attributable, in part, to biases that arise due to post-diagnostic and post-treatment TL measurement. To avoid such biases, we used a Mendelian randomization approach and estimated associations between nine TL-associated SNPs and risk for five common cancer types (breast, lung, colorectal, ovarian and prostate cancer, including subtypes) using data on 51 725 cases and 62 035 controls. We then used an inverse-variance weighted average of the SNP-specific associations to estimate the association between a genetic score representing long TL and cancer risk. The long TL genetic score was significantly associated with increased risk of lung adenocarcinoma ( P = 6.3 x 10 –15 ), even after exclusion of a SNP residing in a known lung cancer susceptibility region ( TERT-CLPTM1L ) P = 6.6 x 10 –6 ). Under Mendelian randomization assumptions, the association estimate [odds ratio (OR) = 2.78] is interpreted as the OR for lung adenocarcinoma corresponding to a 1000 bp increase in TL. The weighted TL SNP score was not associated with other cancer types or subtypes. Our finding that genetic determinants of long TL increase lung adenocarcinoma risk avoids issues with reverse causality and residual confounding that arise in observational studies of TL and disease risk. Under Mendelian randomization assumptions, our finding suggests that longer TL increases lung adenocarcinoma risk. However, caution regarding this causal interpretation is warranted in light of the potential issue of pleiotropy, and a more general interpretation is that SNPs influencing telomere biology are also implicated in lung adenocarcinoma risk.

Description: Numerous genetic factors that influence breast cancer risk are known. However, approximately two-thirds of the overall familial risk remain unexplained. To determine whether some of the missing heritability is due to rare variants conferring high to moderate risk, we tested for an association between the c.5791C〉T nonsense mutation (p.Arg1931*; rs144567652) in exon 22 of FANCM gene and breast cancer. An analysis of genotyping data from 8635 familial breast cancer cases and 6625 controls from different countries yielded an association between the c.5791C〉T mutation and breast cancer risk [odds ratio (OR) = 3.93 (95% confidence interval (CI) = 1.28–12.11; P = 0.017)]. Moreover, we performed two meta-analyses of studies from countries with carriers in both cases and controls and of all available data. These analyses showed breast cancer associations with OR = 3.67 (95% CI = 1.04–12.87; P = 0.043) and OR = 3.33 (95% CI = 1.09–13.62; P = 0.032), respectively. Based on information theory-based prediction, we established that the mutation caused an out-of-frame deletion of exon 22, due to the creation of a binding site for the pre-mRNA processing protein hnRNP A1. Furthermore, genetic complementation analyses showed that the mutation influenced the DNA repair activity of the FANCM protein. In summary, we provide evidence for the first time showing that the common p.Arg1931* loss-of-function variant in FANCM is a risk factor for familial breast cancer.

Description: A new thermodynamic model is presented for calculating phase relations in peridotite, from 0·001 to 60 kbar and from 800°C to liquidus temperatures, in the system NCFMASOCr. This model system is large enough to simulate phase relations and melting of natural peridotite and basaltic liquids. Calculations in the program thermocalc illustrate mantle phase relationships and melting conditions, specifically for the peridotite composition KLB-1. The garnet–spinel transition zone intersects the solidus at 21·4–21·7 kbar, where both Fe 3+ and Cr increase spinel stability, expanding the width of the transition. Orthopyroxene is lost at the solidus at 42 kbar in KLB-1, although this pressure is very sensitive to bulk composition. Calculated oxidation states are in excellent agreement with measured log f O 2 for xenolith suites with mantle Fe 2 O 3 contents in the range 0·1–0·3 wt %. It appears that mantle oxidation state is not just a simple function of P and T , but depends on phase assemblage, and may vary in a complex way within a single assemblage. The liquid model performs well, such that calculated solidus, melt productivity and liquid compositions compare favourably with those of experimental studies, permitting its use in interpolating between, and extrapolating from, experimental P–T conditions. Experimentally challenging but geologically useful regimes can be explored, such as subsolidus samples and very low melt fractions, with application to both mantle xenoliths and the origin of basalt.

Description: The anorthositic members of the Mealy Mountains Intrusive Suite (MMIS; Labrador, Canada) are host to 0·5–5 m diameter pegmatitic, pod-like segregations, originally described as graphic granite pods. U–Pb zircon geochronology confirms that the pods are coeval with the 1650–1630 Ma emplacement age range for the MMIS, yielding ages of 1654 ± 8 to 1628 ± 3·5 Ma. Petrographic and geochemical analysis of five pods from anorthositic rocks of the MMIS reveals that the pods have a diverse compositional range from monzodiorite to granite, varying from Fe-rich and Si-poor, to Fe-poor and Si-rich compositions. Fe-rich, Si-poor pods in the MMIS and other massifs (e.g. Laramie Anorthosite Complex) tend to be hosted by olivine-bearing anorthosites, whereas Si-rich, Fe-poor pods are hosted by pyroxene-bearing anorthosites. Each pod shows a range of graphic, myrmekitic and symplectitic textures, along with distinctive mineral assemblages (e.g. apatite and zircon) and highly enriched trace-element compositions. Evolved mineral assemblages, high concentrations of Fe, Ti and P (and in some cases SiO 2 ), and 10–1000 x chondrite enrichment in light rare earth elements, U, Th and Rb indicate that many of the pods are highly fractionated. The array of textural intergrowths provides clues about the final stages of crystallization in the pods and, by extension, the anorthosites. Macroscopic quartz–K-feldspar graphic intergrowths indicate high-viscosity, fluid-bearing and significantly undercooled magmatic conditions, whereas microscopic myrmekitic (plagioclase–quartz) and symplectitic (plagioclase–orthopyroxene) intergrowths on primary grain boundaries indicate replacement of phases in the presence of reactive fluids. In assessing the nature of these pegmatitic pods based on field, petrographic and geochemical evidence, we conclude that they represent the fluid-bearing, late-stage crystallization products of a residual liquid in the massif anorthosite system. The Fe and Si compositional variations observed in these late-stage pods can be linked to a fundamental olivine–pyroxene dichotomy observed in most Proterozoic anorthosite massifs, suggesting that pulses of magma experience variable contamination (in amount and/or composition) leading to varying differentiation paths. A range of lithologies (monzonites, monzonorites, ferrodiorites and jotunites) observed in similar pod-like structures, as well as dykes and plutons, has been observed in other Proterozoic anorthosite massifs and all have, at one time or another, been interpreted as the residual liquids of anorthosite crystallization. Our observation of in situ pods with similar compositions to all of the aforementioned lithologies, and displaying textures indicative of late-stage crystallization, supports the notion that all of these associated lithologies can be interpreted as comagmatic with, but variably contaminated and isolated residual liquids of, anorthosite crystallization. However, using isotopic evidence we cannot support the notion that the far larger granitic plutons associated with Proterozoic anorthosites are also residual liquids of anorthositic magma fractionation.

Description: We report the results of experiments on two natural marine sediments with different carbonate contents (calcareous clay: CO 2 = 6·1 wt %; marl: CO 2 = 16·2 wt %) at subduction-zone conditions (3 GPa, 750–1200°C). Water (7–15 wt %) was added to the starting materials to simulate the effects of external water addition from within the subducting slab. The onset of melting is at 760°C in water-rich experiments; melt becomes abundant by 800°C. In contrast, the onset of melting in published, water-poor experiments occurs at variable temperatures with the production of significant melt fractions being restricted to more than 900°C (phengite-out). The different solidus temperatures ( T solidus ) can be ascribed to variable fluid X H2O [H 2 O/(CO 2 + H 2 O)], which, in turn, depends on bulk K 2 O, H 2 O and CO 2 . Partial melts in equilibrium with residual garnet, carbonate, quartz/coesite, epidote, rutile, kyanite, phengite, and clinopyroxene are granitic in composition, with substantial dissolved volatiles. Supersolidus runs always contain both silicate melt and solute-rich fluid, indicating that experimental conditions lie below the second critical endpoint in the granite–H 2 O–CO 2 system. Carbonatite melt coexists with silicate melt and solute-rich fluid above 1100°C in the marl. The persistence of carbonate to high temperature, in equilibrium with CO 2 -rich hydrous melts, provides a mechanism to both supply CO 2 to arc magmas and recycle carbon into the deep Earth. The trace element compositions of the experimental glasses constrain the potential contribution of calcareous sediment to arc magmas. The presence of residual epidote and carbonate confers different trace element characteristics when compared with the trace element signal of Ca-poor marine sediments (e.g. pelagic clays). Notably, epidote retains Th and light rare earth elements, such that some melts derived from calcareous sediments have elevated Ba/Th and U/Th, and low La/Sm PUM , thereby resembling fluids conventionally ascribed to altered oceanic crust. Our results emphasize the importance of residual mineralogy, rather than source lithology, in controlling the trace element characteristics of slab-derived fluids.

Description: Three crystal-poor obsidian samples (one dacite, 67 wt % SiO 2 ; two rhyolites, 73 and 75 wt % SiO 2 ), which erupted effusively from monogenetic vents, contain sparse (〈2%) plagioclase phenocrysts that span a remarkably wide and continuous range in composition (≤30 mol % An). Many, but not all, of the plagioclase crystals display diffusion-limited growth textures (e.g. swallow-tails, skeletal, vermiform). Hypotheses to explain the paradox of a wide compositional range despite a low abundance of plagioclase include (1) incorporation of xenocrysts and/or magma mingling, (2) slow crystallization of plagioclase driven by slow cooling in a magma chamber, (3) slow crystallization of plagioclase followed by a resorption (e.g. heating) event, and (4) crystallization driven by rapid degassing (i.e. loss of melt H 2 O) ± rapid cooling during ascent. To test these hypotheses, a series of phase equilibrium experiments were conducted under pure-H 2 O fluid-saturated conditions in a cold-seal pressure vessel between 30 and 300 MPa and 750 and 950°C. The results show that the plagioclase population in each obsidian sample could have grown from their respective melts, with the exception of a single calcic core (An 60–63 ) in one sample. The results additionally rule out slow cooling in a magma chamber, because this would lead to equilibrium abundances of plagioclase (≤20%), which are far higher than what is observed in the samples (〈2%). Nor can resorption (i.e. heating) explain the low abundance of plagioclase, because this would eliminate the more sodic plagioclase crystals and hence the wide compositional range of plagioclase that is observed. The most viable hypothesis is that the sparse plagioclase phenocrysts grew relatively rapidly during magma ascent to the surface; this is consistent with the results of isothermal (850°C) continuous decompression experiments (2·9, 1·0, 0·8, and 0·1 MPa h –1 ), under pure-H 2 O fluid-saturated conditions, which were performed on one of the rhyolites (MLV-36; 73 wt % SiO 2 ) and quenched at P H2O = 89, 58 and 40 MPa. The four decompression rates correspond to degassing rates of 1·6, 0·56, 0·45 and 0·06 wt % H 2 O per day. Decompressions ≥1·0 MPa( P H2O ) h –1 , initiated above the liquidus, quenched to 100% glass at all final P H2O . Decompressions at 0·8 MPa( P H2O ) h –1 , also initiated above the liquidus, led to plagioclase crystals nearly five times larger than those grown in runs decompressed at the same rate, but initiated just below the plagioclase-in curve. It is the kinetic hindrance to nucleation that permits crystal growth to be concentrated on relatively few crystals, leading to larger crystals. Plagioclase growth rates from these experiments show that the largest phenocrysts (~1 mm) in the MLV-36 obsidian could have grown in 〈42 h. A cooling rate of ~1·2°C h –1 closely matches both the increase in melt viscosity with time and the effective undercooling with time that occurs during the 0·8 MPa( P H2O ) h –1 decompression over the first 50 h. The combined results point to crystallization of sparse plagioclase driven by relatively rapid rates of degassing ± cooling during ascent to the surface of melts that were initially above their liquidus. The obsidian samples must have been efficiently segregated as nearly 100% liquids from their respective source regions at H 2 O-fluid undersaturated conditions to attain a degree of superheating upon ascent before reaching fluid saturation.

Description: The Jurassic Vestfjella dyke swarm at the volcanic rifted margin of western Dronning Maud Land represents magmatism related to the incipient Africa–Antarctica rift zone; that is, rift-assemblage magmatism of the Karoo continental flood basalt (CFB) province. Geochemical and Nd–Sr isotopic data for basaltic and picritic dyke samples indicate diverse low-Ti and high-Ti tholeiitic compositions with Nd (180 Ma) ranging from +8 to –17. Combined with previously reported data on a subcategory of ferropicritic dykes, our new data facilitate grouping of the Vestfjella dyke swarm into seven geochemically distinct types. The majority of the dykes exhibit geochemical affinity to continental lithosphere and can be correlated with two previously identified chemical types (CT) of the wall-rock CFB lavas and are accordingly referred to as the CT1 and CT3 dykes. The less abundant Low-Nb and High-Nb dykes, a relatively enriched subtype of CT3 (CT3-E) dykes, and dykes belonging to the depleted and enriched ferropicrite suites represent magma types found only as intrusions. The chemically mid-ocean ridge basalt (MORB)-like Low-Nb and the depleted ferropicrite suite dykes represent, respectively, relatively high- and low-degree partial melting of the same overall depleted mantle (DM)-affinity source in the sublithospheric mantle. In contrast, we ascribe the chemically ocean island basalt (OIB)-like High-Nb dykes and the enriched ferropicrite suite dykes to melting of enriched components in the sublithospheric mantle. Geochemical modelling suggests that the low-Ti affinity CT1 and CT3, and high-Ti affinity CT3-E magma types of Vestfjella dyke may predominantly result from mixing of DM-sourced Low-Nb type magmas with 〈10 wt % of crust- and lithospheric mantle-derived melts. U/Pb zircon dating confirms synchronous emplacement of CT1 dykes and Karoo main-stage CFBs at 182·2 ± 0·9 and 182·2 ± 0·8 Ma, whereas two 40 Ar/ 39 Ar plagioclase plateau ages of 189·2 ± 2·3 Ma (CT1) and 185·5 ± 1·8 Ma (depleted ferropicrite suite), and a mini-plateau age of 186·9 ± 2·8 Ma (CT3-E) for the Vestfjella dykes raise the question of whether the onset of rift-zone magmatism could predate the province-wide c. 179–183 Ma main stage of Karoo magmatism. Notably variable Ca/K spectra suggest that younger 40 Ar/ 39 Ar plagioclase plateau ages of 173, 170, 164, and 154 Ma are related to crystallization of secondary minerals during the late-stage tectono-magmatic development of the Antarctic rifted margin. The occurrence of rare MORB- and OIB-like magma types in Vestfjella and along the African and Antarctic rifted margins suggests melting of geochemically variable depleted and enriched sublithospheric mantle beneath the Africa–Antarctica rift zone. Our models for the Vestfjella dyke swarm indicate that the voluminous lithosphere-affinity low-Ti and high-Ti rift-assemblage tholeiites could have been derived from MORB-like parental magmas by contamination, which implies sublithospheric depleted mantle as the principal source of the CFB magmas of the Africa–Antarctica rift zone.

Description: Monazite laser ablation–split-stream inductively coupled plasma–mass spectrometry (LASS) was used to date monazite in situ in Barrovian-type micaschists of the Moravian zone in the Thaya window, Bohemian Massif. Petrography and garnet zoning combined with pseudosection modelling show that rocks from staurolite–chlorite, staurolite, kyanite and kyanite–sillimanite zones record burial in the S 1 fabric under a moderate geothermal gradient from 4–4·5 kbar and ~530–540°C to 5 kbar and 570°C, 6–7 kbar and 600–640°C, 7·5–8 kbar and 630–650°C, and 8 kbar and 650°C, respectively. In the kyanite and kyanite–sillimanite zones, garnet rim chemistry and local syntectonic replacement of garnet by sillimanite–biotite aggregates point to re-equilibration at 5·5–6 kbar and 630–650°C in the S 2 fabric. Heterogeneously developed retrograde shear zones (S 3 ) are marked by widespread chloritization, but minor chlorite is present in the studied samples. Monazite abundance and size increase with metamorphic grade from 5 µm in the staurolite–chlorite zone to 〉100 µm in the kyanite and kyanite–sillimanite zones. Irrespective of the monazite-forming reaction, this is interpreted as the onset of limited prograde monazite growth at staurolite grade, and continued prograde monazite growth after the kyanite-in reaction, compatible with conditions of about 5·5 kbar and 570°C and 7·5 kbar and 630°C from pseudosection modelling. Monazite is zoned, showing embayments and sharp boundaries between zones, with low Y in the staurolite zone, high-Y cores and low-Y rims in the kyanite zone, and high-Y cores, a low-Y mantle and a high-Y rim in the sillimanite zone. The 207 Pb-corrected 238 U/ 206 Pb ages from three samples range from 344 ± 7 to 330 ± 7 Ma, irrespective of metamorphic grade. The dates from monazite inclusions are interpreted as the ages of the staurolite- and kyanite-in reactions along the prograde path at 340 and 337 ± 7 Ma, respectively. The monazite in the matrix (and some inclusions) is interpreted as dating the prograde crystallization at (340–337) ± 7 Ma within the S 1 fabric, and then being affected by recrystallization at or down to 332 ± 7 Ma in the S 2 and S 3 fabrics. The two groups of data, for 340–337 and 332 Ma, are significantly different when only their in-run uncertainties (±1–3 Myr) are compared and indicate a 9 ± 3 Myr period of monazite (re)crystallization. A systematic increase in heavy rare earth element (HREE) content with decreasing monazite age from 344 to 335 Ma is correlated with growth on the prograde P–T path; the drop in HREE of monazite at 335–328 Ma is assigned to recrystallization. The presence of chlorite even in the least retrogressed samples witnesses limited external fluid availability on the retrograde P–T path. Migration of this fluid was probably responsible for heterogeneous fluid-assisted recrystallization and resetting of original prograde monazite, even where included in garnet, staurolite or kyanite. It is suggested that the rocks passed the chlorite-in reaction on the retrograde path at 332 ± 7 Ma. The timing of burial in the Thaya window, a deformed part of the underthrust Brunia microcontinent, was coeval with exhumation of granulites and migmatites of the Moldanubian orogenic root at c. 340 Ma.

Description: The Fanshan intrusion in the North China Craton (NCC) is concentrically zoned with syenite in the core (Unit 1), surrounded by ultramafic rocks (clinopyroxenite and biotite clinopyroxenite; Unit 2), and an outer rim of garnet-rich clinopyroxenite and orthoclase clinopyroxenite and syenite (Unit 3). The intrusive rocks are composed of variable amounts of Ca-rich augite, biotite, orthoclase, melanite, garnet, magnetite and apatite, with minor primary calcite. Monomineralic apatite rocks, nelsonite and glimmerite exclusively occur in Unit 2. Geochemically, the Fanshan rocks are highly enriched in light rare earth elements (LREE) and large ion lithophile elements (LILE), moderately depleted in high field strength elements (HFSE), and have a limited range of Sr–Nd–O isotopic compositions. The similar mineralogy, mineral compositions, and trace element characteristics of the three units suggest that all the rocks are co-magmatic. The parental magma is ultrapotassic and is akin to kamafugite. Very low-degree partial melting of metasomatized lithospheric mantle best explains the geochemistry and petrogenesis of the parental magmas of the Fanshan intrusion. We propose that the mantle source may have been metasomatized by a hydrous carbonate-bearing melt, which has imprinted the enriched Sr–Nd isotopic signature and incompatible element enrichment with conspicuous negative Nb–Ta–Zr–Hf–Ti anomalies and LREE enrichments. The mantle source enrichment may be correlated with oceanic sediment recycling during southward subduction of the Paleo-Asian oceanic plate during the Carboniferous and Permian. We propose that crystal settling and mechanical sorting combined with repeated primitive magma replenishment and mixing with previously fractionated magma is the predominant process responsible for the formation of the apatite ores.

Description: Experimental studies of mantle petrology find that small concentrations of water and carbon dioxide have a large effect on the solidus temperature and distribution of melting in the upper mantle. However, it has remained unclear what effect small fractions of deep, volatile-rich melts have on melt transport and reactive melting in the shallow asthenosphere. Here we present theory and computations indicating that low-degree, reactive, volatile-rich melts cause channelization of magmatic flow at depths approximately corresponding to the anhydrous solidus temperature. These results are obtained with a novel method to simulate the thermochemical evolution of the upper mantle in the presence of volatiles. The method uses a thermodynamically consistent framework for reactive, disequilibrium, multi-component melting. It is coupled with a system of equations representing conservation of mass, momentum, and energy for a partially molten grain aggregate. Application of this method in two-phase, three-component upwelling-column models demonstrates that it reproduces leading-order features of hydrated and carbonated peridotite melting; in particular, it captures the production of low-degree, volatile-rich melt at depths far below the volatile-free solidus. The models predict that segregation of volatile-rich, deep melts promotes a reactive channelling instability that creates fast and chemically isolated pathways of melt extraction. Reactive channelling occurs where volatile-rich melts flux the base of the silicate melting region, enhancing dissolution of fusible components from the ambient mantle. We find this effect to be similarly expressed for models of both hydrated and carbonated mantle melting. These findings indicate that despite their small concentrations, water and carbon dioxide have an important control on the extent and style of magma genesis, as well as on the dynamics of melt transport.

Description: A study of whole-rock major and trace element and Sr–Nd–Pb–Hf isotope compositions, combined with zircon U–Pb ages and Hf–O isotopes, for postcollisional intermediate volcanic rocks from the Dabie orogen, China provides constraints on the origin of andesitic magmas and insights into slab–mantle interaction in continental subduction channels. The volcanic rocks exhibit variable contents of SiO 2 (50·28–63·86 wt %), MgO (1·18–4·65 wt %), (Fe 2 O 3 ) T (3·60–8·53 wt %), Al 2 O 3 (12·92–18·95 wt %), Na 2 O (2·08–6·30 wt %) and K 2 O (0·73–5·25 wt %). They are mainly trachyandesites, with lesser amounts of basaltic trachyandesite, andesite, dacite and trachyte, characterized by subduction-like trace element distribution patterns showing enrichment of large ion lithophile elements and light rare earth elements but depletion of high field strength elements. The volcanic rocks have relatively enriched Sr–Nd–Pb–Hf isotope compositions, with high initial 87 Sr/ 86 Sr ratios of 0·7075–0·7110, highly negative Nd ( t ) values of –23·1 to –15·0, Hf ( t ) values of –29·8 to –18·3 and elevated 207 Pb/ 204 Pb and 208 Pb/ 204 Pb ratios at given 206 Pb/ 204 Pb ratios. Zircon Hf–O isotope analyses yield negative Hf ( t ) values of –31·0 to –17·8 and 18 O values of 4·4–6·8 for syn-magmatic domains. Zircon U–Pb dating yields consistent Early Cretaceous ages of 124 ± 3 to 130 ± 2 Ma for magma emplacement. Residual zircon cores yield Triassic, Neoproterozoic and Paleoproterozoic U–Pb ages, consistent with the ages of tectonothermal events involving ultrahigh-pressure metamorphism and protolith formation in the Dabie orogen. The zircon cores also yield a few low to negative 18 O values. An integrated interpretation of these geochemical characteristics is that the andesitic magmas were derived by partial melting of metasomatized zones in the orogenic lithospheric mantle. The metasomatites were generated by reaction of subcontinental lithospheric mantle wedge peridotite with felsic melts that originated from deeply subducted continental crust during continental collision in the Triassic. Melt–peridotite reaction in a subduction channel is therefore a key to the origin of the mantle sources of andesitic magmas in collisional orogens.

Description: The late Proterozoic Ntaka Ultramafic Complex is a body of dominantly pyroxenitic cumulate rocks containing cyclic alternations of olivine–orthopyroxene cumulates. Chemical zoning in the pyroxenes has been imaged at 25–40 µm resolution using desktop microbeam X-ray fluorescence mapping followed up with laser ablation–inductively coupled plasma mass spectrometry analysis for minor and trace elements on selected samples. Poikilitic and granular harzburgites are finely intermingled, in some cases on a centimetre scale in the same thin section. Poikilitic varieties display spectacular textures, ranging from isolated equant orthopyroxene oikocrysts within olivine-rich heteradcumulate harzburgites to rocks composed entirely of interlocking centimetre-sized anhedral orthopyroxene oikocrysts containing sharply bounded idiomorphic Cr-enriched cores. The poikilitic harzburgites are interlayered with cumulate pyroxenites in which orthopyroxene grains show a variety of zoning patterns: Cr-rich cores similar to those in the oikocrysts; sharply bounded oscillatory zoned cores; and reverse zoning with Cr-poor cores and Cr-enriched rims. A further variation is the presence of a mingled harzburgite lithology in which dunite or poikilitic harzburgite is invaded on a centimetre scale by diffuse vein networks or patches of coarse orthopyroxenite. This range of textures and lithologies attests to a more complex set of processes than implied by the standard cumulus theory model in which oikocrysts are considered to have crystallized from intercumulus liquid within a permeable crystal mush. A range of hypotheses is proposed, including infiltration metasomatism of original olivine cumulates by migrating orthopyroxene-saturated pore fluid; however, the textural relationships, whole-rock chemistry and Cr zoning within the grains can best be explained by a model in which the orthopyroxene oikocrysts form in part or whole as mechanically accumulated cumulus grains. The complexity of zoning patterns is attributed to stirring of entrained olivine and orthopyroxene crystals within a heterogeneous flowing crystal mush, where the transporting magma has a wide range of silica contents owing to poorly stirred incorporation of siliceous country-rock material. The Cr-rich orthopyroxenite component grew from Si-enriched chromite-saturated magma. Mingled lithologies developed after accumulation as a result of percolation and infiltration metasomatism by Si-enriched liquid derived by melting of xenoliths within the crystal pile. The model may be more generally applicable: dunite–harzburgite cycles, common in many layered intrusions, may reflect variable degrees of contamination rather than cycles of fractional crystallization and replenishment.

Description: New thermodynamic data for skiagite garnet (Fe 3 Fe 2 3+ Si 3 O 12 ) are derived from experimental phase-equilibrium data that extend to 10 GPa and are applied to oxybarometry of mantle peridotites using a revised six-component garnet mixing model. Skiagite is more stable by 12 kJ mol –1 than in a previous calibration of the equilibrium 2 skiagite = 4 fayalite + ferrosilite + O 2 , and this leads to calculated oxygen fugacities that are higher (more oxidized) by around 1–1·5 logfO2 units. A new calculation method and computer program incorporates four independent oxybarometers (including 2 pyrope + 2 andradite + 2 ferrosilite = 2 grossular + 4 fayalite + 3 enstatite + O 2 ) for use on natural peridotite samples to yield optimum logfO2 estimates by the method of least squares. These estimates should be more robust than those based on any single barometer and allow assessment of possible disequilibrium in assemblages. A new set of independent oxybarometers for spinel-bearing peridotites is also presented here, including a new reaction 2 magnetite + 3 enstatite = 3 fayalite + 3 forsterite + O 2 . These recalibrations combined with internally consistent PT determinations for published analyses of mantle peridotites with analysed Fe 2 O 3 data for garnets, from both cratonic (Kaapvaal, Siberia and Slave) and circumcratonic (Baikal Rift) regions, provide revised estimates of oxidation state in the lithospheric mantle. Estimates of logfO2 for spinel assemblages are more reduced than those based on earlier calibrations, whereas garnet-bearing assemblages are more oxidized. Importantly, this lessens considerably the difference between garnet and spinel oxybarometry that was observed with previous published calibrations.

Description: The Kidnappers [~1200 km 3 dense rock equivalent (DRE)] and Rocky Hill (~200 km 3 DRE) caldera-forming events in the Taupo Volcanic Zone, New Zealand, were erupted in close succession from the Mangakino volcanic centre. They have identical radiometric ages at ~1 Ma, yet erosion along the contact between the two deposits suggests that some years to decades separated the two eruptions. Field constraints and the similarities of crystal textures and compositions and glass chemistries of both eruption deposits demonstrate that they came from one overall magmatic system with a common crystal mush source. However, second-order variations in these parameters confirm that the Kidnappers and Rocky Hill deposits represent distinct events and are not the products of a single zoned magma chamber. The systematically zoned Kidnappers fall deposits provide evidence for the tapping of three discrete magma bodies, whereas the succeeding Kidnappers ignimbrite is compositionally more diverse. The transition from fall to flow deposition marks a change in the style of caldera collapse and the simultaneous evacuation of discrete but compositionally diverse melts, each of which underwent a distinct evolution and was held at slightly different P–T conditions prior to eruption. Contrasting plagioclase and orthopyroxene zonation patterns are present in pumices originating from three discrete magma bodies. Less evolved mafic melts interacted with the system, which mobilized portions of the final erupted melt through heating and volatile or chemical exchange in the mush. The two largest Kidnappers melt-dominant bodies were re-tapped in modified form, or re-established from their common mush source, prior to the Rocky Hill event. Rocky Hill pumices contain common, fluid-affected antecrystic crystal clots derived from chamber wall material. Amphibole compositions from each eruption reflect melt evolution processes and, in particular, the contemporaneous crystallization of biotite and breakdown of orthopyroxene. Plagioclase and orthopyroxene from Rocky Hill pumices share common zonation patterns with those from the two largest magma bodies in the Kidnappers. The rapid production of new melt-dominant bodies and the triggering of the Rocky Hill eruption reflect the ability of the magmatic system to rejuvenate on a geologically short timescale. The Mangakino centre did not follow a typical cycle of decreased activity after the supervolcanic Kidnappers event, instead producing a second caldera-forming eruption, within years to decades from the same system.

Description: Ambrym, a basaltic volcano in the Vanuatu Arc, has displayed variable eruptive behaviour throughout the past century, with major eruptions occurring both on the volcano flanks and at multiple vents within its caldera. These have been interspersed with periods of relative quiescence marked by extensive passive degassing at active, intra-caldera lava lakes, which experience occasional Strombolian explosions. Volcanic rocks from all vents and eruptive styles display similar isotope and incompatible trace element compositions, suggesting that all are derived from the same primary melt by fractional crystallization. Major eruptions are commonly responsible for effusion of the least evolved lavas examined (SiO 2 ~ 50 wt %; MgO ~ 5 wt %). Although all are geochemically similar, petrological differences discriminate between lavas erupted during flank and intra-caldera eruptions. Phyric basalts with homogeneous mineral compositions are common to flank eruptions, whereas crystal-rich basalts with variable mineral compositions, many not in equilibrium with their host liquid, are a feature of intra-caldera lavas. Lava lake samples are slightly more evolved than those from effusive eruptions (SiO 2 ~ 51–52 wt %; MgO ~ 4 wt %), as a result of additional crystallization during periods of relative quiescence. The diverse petrology of the intra-caldera lavas can be explained by mixing of replenishment magmas similar to those erupted from the volcano flanks with residual magma from lava lake activity. Flank eruptions exploit dykes that bypass the shallow reservoir involved with lava lake activity, limiting their interaction with this component.