ALBERT

Description: The Vesicle-associated membrane protein ( V AMP)- A ssociated P rotein B (VAPB) is the causative gene of amyotrophic lateral sclerosis 8 (ALS8) in humans. Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by selective death of motor neurons leading to spasticity, muscle atrophy and paralysis. VAP proteins have been implicated in various cellular processes, including intercellular signalling, synaptic remodelling, lipid transport and membrane trafficking and yet, the molecular mechanisms underlying ALS8 pathogenesis remain poorly understood. We identified the conserved phosphoinositide phosphatase Sac1 as a Drosophila VAP (DVAP)-binding partner and showed that DVAP is required to maintain normal levels of phosphoinositides. Downregulating either Sac1 or DVAP disrupts axonal transport, synaptic growth, synaptic microtubule integrity and the localization of several postsynaptic components. Expression of the disease-causing allele ( DVAP-P58S ) in a fly model for ALS8 induces neurodegeneration, elicits synaptic defects similar to those of DVAP or Sac1 downregulation and increases phosphoinositide levels. Consistent with a role for Sac1-mediated increase of phosphoinositide levels in ALS8 pathogenesis, we found that Sac1 downregulation induces neurodegeneration in a dosage-dependent manner. In addition, we report that Sac1 is sequestered into the DVAP-P58S-induced aggregates and that reducing phosphoinositide levels rescues the neurodegeneration and suppresses the synaptic phenotypes associated with DVAP-P58S transgenic expression. These data underscore the importance of DVAP–Sac1 interaction in controlling phosphoinositide metabolism and provide mechanistic evidence for a crucial role of phosphoinositide levels in VAP-induced ALS.

Description: The pubertal height growth spurt is a distinctive feature of childhood growth reflecting both the central onset of puberty and local growth factors. Although little is known about the underlying genetics, growth variability during puberty correlates with adult risks for hormone-dependent cancer and adverse cardiometabolic health. The only gene so far associated with pubertal height growth, LIN28B, pleiotropically influences childhood growth, puberty and cancer progression, pointing to shared underlying mechanisms. To discover genetic loci influencing pubertal height and growth and to place them in context of overall growth and maturation, we performed genome-wide association meta-analyses in 18 737 European samples utilizing longitudinally collected height measurements. We found significant associations ( P 〈 1.67 x 10 –8 ) at 10 loci, including LIN28B . Five loci associated with pubertal timing, all impacting multiple aspects of growth. In particular, a novel variant correlated with expression of MAPK3 , and associated both with increased prepubertal growth and earlier menarche. Another variant near ADCY3-POMC associated with increased body mass index, reduced pubertal growth and earlier puberty. Whereas epidemiological correlations suggest that early puberty marks a pathway from rapid prepubertal growth to reduced final height and adult obesity, our study shows that individual loci associating with pubertal growth have variable longitudinal growth patterns that may differ from epidemiological observations. Overall, this study uncovers part of the complex genetic architecture linking pubertal height growth, the timing of puberty and childhood obesity and provides new information to pinpoint processes linking these traits.

Description: Visual refractive errors (REs) are complex genetic traits with a largely unknown etiology. To date, genome-wide association studies (GWASs) of moderate size have identified several novel risk markers for RE, measured here as mean spherical equivalent (MSE). We performed a GWAS using a total of 7280 samples from five cohorts: the Age-Related Eye Disease Study (AREDS); the KORA study (‘Cooperative Health Research in the Region of Augsburg’); the Framingham Eye Study (FES); the Ogliastra Genetic Park-Talana (OGP-Talana) Study and the Multiethnic Study of Atherosclerosis (MESA). Genotyping was performed on Illumina and Affymetrix platforms with additional markers imputed to the HapMap II reference panel. We identified a new genome-wide significant locus on chromosome 16 (rs10500355, P = 3.9 x 10 –9 ) in a combined discovery and replication set (26 953 samples). This single nucleotide polymorphism (SNP) is located within the RBFOX1 gene which is a neuron-specific splicing factor regulating a wide range of alternative splicing events implicated in neuronal development and maturation, including transcription factors, other splicing factors and synaptic proteins.

Description: Small intestinal epithelial cells (sIECs) have a significant share in whole body metabolism as they perform enzymatic digestion and absorption of nutrients. Furthermore, the diet plays a key role in a number of complex diseases including obesity and diabetes. The impact of diet and altered genetic backgrounds on human metabolism may be studied by using computational modeling. A metabolic reconstruction of human sIECs was manually assembled using the literature. The resulting sIEC model was subjected to two different diets to obtain condition-specific metabolic models. Fifty defined metabolic tasks evaluated the functionalities of these models, along with the respective secretion profiles, which distinguished between impacts of different dietary regimes. Under the average American diet, the sIEC model resulted in higher secretion flux for metabolites implicated in metabolic syndrome. In addition, enzymopathies were analyzed in the context of the sIEC metabolism. Computed results were compared with reported gastrointestinal (GI) pathologies and biochemical defects as well as with biomarker patterns used in their diagnosis. Based on our simulations, we propose that (i) sIEC metabolism is perturbed by numerous enzymopathies, which can be used to study cellular adaptive mechanisms specific for such disorders, and in the identification of novel co-morbidities, (ii) porphyrias are associated with both heme synthesis and degradation and (iii) disturbed intestinal gamma-aminobutyric acid synthesis may be linked to neurological manifestations of various enzymopathies. Taken together, the sIEC model represents a comprehensive, biochemically accurate platform for studying the function of sIEC and their role in whole body metabolism.

Description: Large intronic expansions of the triplet-repeat sequence (GAA.TTC) cause transcriptional repression of the Frataxin gene ( FXN ) leading to Friedreich's ataxia (FRDA). We previously found that GAA-triplet expansions stimulate heterochromatinization in vivo in transgenic mice. We report here using chromosome conformation capture (3C) coupled with high-throughput sequencing that the GAA-repeat expansion in FRDA cells stimulates a higher-order structure as a fragment containing the GAA-repeat expansion showed an increased interaction frequency with genomic regions along the FXN locus. This is consistent with a more compacted chromatin and coincided with an increase in both constitutive H3K9me3 and facultative H3K27me3 heterochromatic marks in FRDA. Consistent with this, DNase I accessibility in regions flanking the GAA repeats in patients was decreased compared with healthy controls. Strikingly, this effect could be antagonized with the class III histone deactylase (HDAC) inhibitor vitamin B3 (nicotinamide) which activated the silenced FXN gene in several FRDA models. Examination of the FXN locus revealed a reduction of H3K9me3 and H3K27me3, an increased accessibility to DNase I and an induction of euchromatic H3 and H4 histone acetylations upon nicotinamide treatment. In addition, transcriptomic analysis of nicotinamide treated and untreated FRDA primary lymphocytes revealed that the expression of 67% of genes known to be dysregulated in FRDA was ameliorated by the treatment. These findings show that nictotinamide can up-regulate the FXN gene and reveal a potential mechanism of action for nicotinamide in reactivating the epigenetically silenced FXN gene and therefore support the further assessment of HDAC inhibitors (HDACi's) in FRDA and diseases caused by a similar mechanism.

Description: Neuronal ceroid lipofuscinosis (NCL), commonly referred to as Batten disease, is a group of autosomal recessive neurodegenerative diseases of childhood characterized by seizures, blindness, motor and cognitive decline and premature death. Currently, there are over 400 known mutations in 14 different genes, leading to five overlapping clinical variants of NCL. A large portion of these mutations lead to premature stop codons (PTCs) and are predicted to predispose mRNA transcripts to nonsense-mediated decay (NMD). Nonsense-mediated decay is associated with a number of other genetic diseases and is an important regulator of disease pathogenesis. We contend that NMD targets PTCs in NCL gene transcripts for degradation. A number of PTC mutations in CLN1 , CLN2 and CLN3 lead to a significant decrease in mRNA transcripts and a corresponding decrease in protein levels and function in patient-derived lymphoblast cell lines. Inhibiting NMD leads to an increased transcript level, and where protein function is known, increased activity. Treatment with read-through drugs also leads to increased protein function. Thus, NMD provides a promising therapeutic target that would allow read-through of transcripts to enhance protein function and possibly ameliorate Batten disease pathogenesis.

Description: Otitis media with effusion (OME) is the most common cause of hearing loss in children and tympanostomy to alleviate the condition remains the commonest surgical intervention in children in the developed world. Chronic and recurrent forms of OM are known to have a very significant genetic component, however, until recently little was known of the underlying genes involved. The identification of mouse models of chronic OM has indicated a role of transforming growth factor beta (TGFβ) signalling and its impact on responses to hypoxia in the inflamed middle ear. We have, therefore, investigated the role of TGFβ signalling and identified and characterized a new model of chronic OM carrying a mutation in the gene for transforming growth interacting factor 1 ( Tgif1 ). Tgif1 homozygous mutant mice have significantly raised auditory thresholds due to a conductive deafness arising from a chronic effusion starting at around 3 weeks of age. The OM is accompanied by a significant thickening of the middle ear mucosa lining, expansion of mucin-secreting goblet cell populations and raised levels of vascular endothelial growth factor, TNF-α and IL-1β in ear fluids. We also identified downstream effects on TGFβ signalling in middle ear epithelia at the time of development of chronic OM. Both phosphorylated SMAD2 and p21 levels were lowered in the homozygous mutant, demonstrating a suppression of the TGFβ pathway. The identification and characterization of the Tgif mutant supports the role of TGFβ signalling in the development of chronic OM and provides an important candidate gene for genetic studies in the human population.

Description: Mutations in the epithelial cell adhesion molecule (EpCAM; CD326) gene are causal for congenital tufting enteropathy (CTE), a disease characterized by intestinal abnormalities resulting in lethal diarrhea in newborns. Why the different mutations all lead to the same disease is not clear. Here, we report that most mutations, including a novel intronic variant, will result in lack of EpCAM's transmembrane domain, whereas two mutations allow transmembrane localization. We find that these mutants are not routed to the plasma membrane, and that truncated mutants are secreted or degraded. Thus, all epcam mutations lead to loss of cell-surface EpCAM, resulting in CTE.

Description: Rett syndrome (RTT), an X-linked postnatal disorder, results from mutations in Methyl CpG-binding protein 2 ( MECP2 ). Survival and breathing in Mecp2 NULL/Y animals are improved by an N-terminal tripeptide of insulin-like growth factor I (IGF-I) treatment. We determined that Mecp2 NULL/Y animals also have a metabolic syndrome and investigated whether IGF-I treatment might improve this phenotype. Mecp2 NULL/Y mice were treated with a full-length IGF-I modified with the addition of polyethylene glycol (PEG-IGF-I), which improves pharmacological properties. Low-dose PEG-IGF-I treatment slightly improved lifespan and heart rate in Mecp2 NULL/Y mice; however, high-dose PEG-IGF-I decreased lifespan. To determine whether insulinotropic off-target effects of PEG-IGF-I caused the detrimental effect, we treated Mecp2 NULL/Y mice with insulin, which also decreased lifespan. Thus, the clinical benefit of IGF-I treatment in RTT may critically depend on the dose used, and caution should be taken when initiating clinical trials with these compounds because the beneficial therapeutic window is narrow.

Description: Abnormal metabolism of the tau protein is central to the pathogenesis of a number of dementias, including Alzheimer's disease. Aberrant alternative splicing of exon 10 in the tau pre-mRNA resulting in an imbalance of tau isoforms is one of the molecular causes of the inherited tauopathy, FTDP-17. We showed previously in heterologous systems that exon 10 inclusion in tau mRNA could be modulated by spliceosome-mediated RNA trans -splicing (SMaRT). Here, we evaluated the potential of trans -splicing RNA reprogramming to correct tau mis-splicing in differentiated neurons in a mouse model of tau mis-splicing, the htau transgenic mouse line, expressing the human MAPT gene in a null mouse Mapt background. Trans -splicing molecules designed to increase exon 10 inclusion were delivered to neurons using lentiviral vectors. We demonstrate reprogramming of tau transcripts at the RNA level after transduction of cultured neurons or after direct delivery and long-term expression of viral vectors into the brain of htau mice in vivo . Tau RNA trans -splicing resulted in an increase in exon 10 inclusion in the mature tau mRNA. Importantly, we also show that the trans -spliced product is translated into a full-length chimeric tau protein. These results validate the potential of SMaRT to correct tau mis-splicing and provide a framework for its therapeutic application to neurodegenerative conditions linked to aberrant RNA processing.

Description: Proper function of the motor unit is dependent upon the correct development of dendrites and axons. The infant/childhood onset motoneuron disease spinal muscular atrophy (SMA), caused by low levels of the survival motor neuron (SMN) protein, is characterized by muscle denervation and paralysis. Although different SMA models have shown neuromuscular junction defects and/or motor axon defects, a comprehensive analysis of motoneuron development in vivo under conditions of low SMN will give insight into why the motor unit becomes dysfunctional. We have generated genetic mutants in zebrafish expressing low levels of SMN from the earliest stages of development. Analysis of motoneurons in these mutants revealed motor axons were often shorter and had fewer branches. We also found that motoneurons had significantly fewer dendritic branches and those present were shorter. Analysis of motor axon filopodial dynamics in live embryos revealed that mutants had fewer filopodia and their average half-life was shorter. To determine when SMN was needed to rescue motoneuron development, SMN was conditionally induced in smn mutants during embryonic stages. Only when SMN was added back soon after motoneurons were born, could later motor axon development be rescued. Importantly, analysis of motor behavior revealed that animals with motor axon defects had significant deficits in motor output. We also show that SMN is required earlier for motoneuron development than for survival. These data support that SMN is needed early in development of motoneuron dendrites and axons to develop normally and that this is essential for proper connectivity and movement.

Description: Environmental factors including ionizing radiation and chemical agents have been known to be able to induce DNA rearrangements and cause genomic structural variations (SVs); however, the roles of intrinsic characteristics of the human genome, such as regional genome architecture, in SV formation and the potential mechanisms underlying genomic instability remain to be further elucidated. Recently, locus-specific observations showed that ‘self-chain’ (SC), a group of short low-copy repeats (LCRs) in the human genome, can induce autism-associated SV mutations of the MECP2 and NRXN1 genes. In this study, we conducted a genome-wide analysis to investigate SCs and their potential roles in genomic SV formation. Utilizing a vast amount of human SV data, we observed a significant biased distribution of human germline SV breakpoints to SC regions. Notably, the breakpoint distribution pattern is different between SV types across deletion, duplication, inversion and insertion. Our observations were coincident with a mechanism of SC-induced DNA replicative errors, whereas SC may sporadically be used as substrates of nonallelic homologous recombination (NAHR). This contention was further supported by our consistent findings in somatic SV mutations of cancer genomes, suggesting a general mechanism of SC-induced genome instability in human germ and somatic cells.

Description: Mowat–Wilson syndrome (MWS) is a severe intellectual disability (ID)-distinctive facial gestalt-multiple congenital anomaly syndrome, commonly associating microcephaly, epilepsy, corpus callosum agenesis, conotruncal heart defects, urogenital malformations and Hirschsprung disease (HSCR). MWS is caused by de novo heterozygous mutations in the ZEB2 gene. The majority of mutations lead to haplo-insufficiency through premature stop codons or large gene deletions. Only three missense mutations have been reported so far; none of which resides in a known functional domain of ZEB2. In this study, we report and analyze the functional consequences of three novel missense mutations, p.Tyr1055Cys, p.Ser1071Pro and p.His1045Arg, identified in the highly conserved C-zinc-finger (C-ZF) domain of ZEB2 . Patients' phenotype included the facial gestalt of MWS and moderate ID, but no microcephaly, heart defects or HSCR. In vitro studies showed that all the three mutations prevented binding and repression of the E-cadherin promoter, a characterized ZEB2 target gene. Taking advantage of the zebrafish morphant technology, we performed rescue experiments using wild-type (WT) and mutant human ZEB2 mRNAs. Variable, mutation-dependent, embryo rescue, correlating with the severity of patients' phenotype, was observed. Our data provide evidence that these missense mutations cause a partial loss of function of ZEB2, suggesting that its role is not restricted to repression of E-cadherin. Functional domains other than C-ZF may play a role in early embryonic development. Finally, these findings broaden the clinical spectrum of ZEB2 mutations, indicating that MWS ought to be considered in patients with lesser degrees of ID and a suggestive facial gestalt, even in the absence of congenital malformation.

Description: Mutations in the gene encoding Fused in Sarcoma ( FUS ) cause amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder. FUS is a predominantly nuclear DNA- and RNA-binding protein that is involved in RNA processing. Large FUS-immunoreactive inclusions fill the perikaryon of surviving motor neurons of ALS patients carrying mutations at post-mortem. This sequestration of FUS is predicted to disrupt RNA processing and initiate neurodegeneration. Here, we demonstrate that C-terminal ALS mutations disrupt the nuclear localizing signal (NLS) of FUS resulting in cytoplasmic accumulation in transfected cells and patient fibroblasts. FUS mislocalization is rescued by the addition of the wild-type FUS NLS to mutant proteins. We also show that oxidative stress recruits mutant FUS to cytoplasmic stress granules where it is able to bind and sequester wild-type FUS. While FUS interacts with itself directly by protein–protein interaction, the recruitment of FUS to stress granules and interaction with PABP are RNA dependent. These findings support a two-hit hypothesis, whereby cytoplasmic mislocalization of FUS protein, followed by cellular stress, contributes to the formation of cytoplasmic aggregates that may sequester FUS, disrupt RNA processing and initiate motor neuron degeneration.

Description: Genome-wide association studies (GWASs) have identified multiple common genetic variants associated with an increased risk of testicular germ cell tumors (TGCTs). A previous GWAS reported a possible TGCT susceptibility locus on chromosome 1q23 in the UCK2 gene, but failed to reach genome-wide significance following replication. We interrogated this region by conducting a meta-analysis of two independent GWASs including a total of 940 TGCT cases and 1559 controls for 122 single-nucleotide polymorphisms (SNPs) on chromosome 1q23 and followed up the most significant SNPs in an additional 2202 TGCT cases and 2386 controls from four case–control studies. We observed genome-wide significant associations for several UCK2 markers, the most significant of which was for rs3790665 ( P Combined = 6.0 x 10 –9 ). Additional support is provided from an independent familial study of TGCT where a significant over-transmission for rs3790665 with TGCT risk was observed ( P FBAT = 2.3 x 10 –3 ). Here, we provide substantial evidence for the association between UCK2 genetic variation and TGCT risk.

Description: The TP53 tumor suppressor pathway is abrogated by TP53 mutations in the majority of human cancers. Increased levels of wild-type TP53 in aggressive neuroblastomas appear paradox but are tolerated by tumor cells due to co-activation of the TP53 ubiquitin ligase, MDM2. The role of the MDM2 antagonist, p14 ARF , in controlling the TP53-MDM2 balance in neuroblastoma is unresolved. In the present study, we show that conditional p14 ARF expression substantially suppresses viability, clonogenicity and anchorage-independent growth in p14 ARF -deficient or MYCN -amplified neuroblastoma cell lines. Furthermore, ectopic 14 ARF expression induced accumulation of cells in the G1 phase and apoptosis, which was paralleled by accumulation of TP53 and its targets. Comparative genomic hybridization analysis of 193 primary neuroblastomas detected one homozygous deletion of CDKN2A (encoding both p14 ARF and p16 INK4A ) and heterozygous loss of CDKN2A in 22% of tumors. Co-expression analysis of p14 ARF and its transactivator, E2F1, in a set of 68 primary tumors revealed only a weak correlation, suggesting that further regulatory mechanisms govern p14 ARF expression in neuroblastomas. Intriguingly, analyses utilizing chromatin immunoprecipitation revealed different histone mark-defined epigenetic activity states of p14 ARF in neuroblastoma cell lines that correlated with endogenous p14 ARF expression but not with episomal p14 ARF promoter reporter activity, indicating that the native chromatin context serves to epigenetically repress p14 ARF in neuroblastoma cells. Collectively, the data pinpoint p14 ARF as a critical factor for efficient TP53 response in neuroblastoma cells and assign p14 ARF as a neuroblastoma suppressor candidate that is impaired by genomic loss and epigenetic repression.

Description: Tuberous sclerosis complex (TSC) is a genetic disorder caused by mutations in TSC1 or TSC2 resulting in hyperactivity of the mammalian target of rapamycin and disabling brain lesions. These lesions contain misplaced neurons enriched in hypoxia-inducible factor 1a (HIF1a). However, the relationship between TSC1/2 and HIF1a and the function of HIF1a in TSC neurons remain unexplored. Here, we examine the degree of HIF1a activity and its function in newborn Tsc1 null neurons in a mouse model of TSC. Using single cell electroporation in the neurogenic subventricular zone (SVZ) of neonatal mice, we deleted Tsc1 and generated olfactory lesions containing misplaced Tsc1 null neurons as previously reported. These newborn neurons displayed elevated HIF1a-mediated transcriptional activity when compared with Tsc1 heterozygote neurons and a marked resistance to cell death induced by a HIF1a antagonist. Electroporation of Hif1a targeting short hairpin RNA (shRNA) or dominant negative HIF1a constructs resulted in 80–90% loss of Tsc1 null newborn neurons although sparing SVZ stem cells. Consistent with this later finding, induction of Hif1a shRNA expression during synaptic integration thus bypassing neuron production also resulted in newborn neuron death. Collectively, these results suggest that HIF1a acts as a molecular determinant of newborn neuron survival and that its TSC1-dependent up-regulation gave Tsc1 null neurons a survival advantage, despite their misplacement in a novel microenvironment.

Description: Several known or putative glycosyltransferases are required for the synthesis of laminin-binding glycans on alpha-dystroglycan (αDG), including POMT1, POMT2, POMGnT1, LARGE, Fukutin, FKRP, ISPD and GTDC2. Mutations in these glycosyltransferase genes result in defective αDG glycosylation and reduced ligand binding by αDG causing a clinically heterogeneous group of congenital muscular dystrophies, commonly referred to as dystroglycanopathies. The most severe clinical form, Walker–Warburg syndrome (WWS), is characterized by congenital muscular dystrophy and severe neurological and ophthalmological defects. Here, we report two homozygous missense mutations in the β-1,3- N -acetylglucosaminyltransferase 1 ( B3GNT1 ) gene in a family affected with WWS. Functional studies confirmed the pathogenicity of the mutations. First, expression of wild-type but not mutant B3GNT1 in human prostate cancer (PC3) cells led to increased levels of αDG glycosylation. Second, morpholino knockdown of the zebrafish b3gnt1 orthologue caused characteristic muscular defects and reduced αDG glycosylation. These functional studies identify an important role of B3GNT1 in the synthesis of the uncharacterized laminin-binding glycan of αDG and implicate B3GNT1 as a novel causative gene for WWS.

Description: The transcription factor Wilms' tumor gene 1, WT1, is implicated both in normal developmental processes and in the generation of a variety of solid tumors and hematological malignancies. Physical interactions of other cellular proteins with WT1 are known to modulate its function. We previously identified the Krüppel-like zinc-finger protein, ZNF224, as a novel human WT1-associating protein that enhances the transcriptional activation of the human vitamin D receptor promoter by WT1. Here, we have analyzed the effects of WT1–ZNF224 interaction on the expression of apoptosis-regulating genes in the chronic myelogenous leukemia (CML) K562 cell line. The results demonstrated that ZNF224 acts in fine tuning of WT1-dependent control of gene expression, acting as a co-activator of WT1 in the regulation of proapoptotic genes and suppressing WT1 mediated transactivation of antiapoptotitc genes. Moreover, the DNA damaging drug cytosine arabinoside (ara-C) induces expression of ZNF224 in K562 cells and this induction enhances cell apoptotic response to ara-C. These findings suggest that ZNF224 can be a mediator of DNA damage-induced apoptosis in leukemia cells.

Description: Charcot–Marie–Tooth disease type 1C (CMT1C) is a dominantly inherited motor and sensory neuropathy. Despite human genetic evidence linking missense mutations in SIMPLE to CMT1C, the in vivo role of CMT1C-linked SIMPLE mutations remains undetermined. To investigate the molecular mechanism underlying CMT1C pathogenesis, we generated transgenic mice expressing either wild-type or CMT1C-linked W116G human SIMPLE. Mice expressing mutant, but not wild type, SIMPLE develop a late-onset motor and sensory neuropathy that recapitulates key clinical features of CMT1C disease. SIMPLE mutant mice exhibit motor and sensory behavioral impairments accompanied by decreased motor and sensory nerve conduction velocity and reduced compound muscle action potential amplitude. This neuropathy phenotype is associated with focally infolded myelin loops that protrude into the axons at paranodal regions and near Schmidt–Lanterman incisures of peripheral nerves. We find that myelin infolding is often linked to constricted axons with signs of impaired axonal transport and to paranodal defects and abnormal organization of the node of Ranvier. Our findings support that SIMPLE mutation disrupts myelin homeostasis and causes peripheral neuropathy via a combination of toxic gain-of-function and dominant-negative mechanisms. The results from this study suggest that myelin infolding and paranodal damage may represent pathogenic precursors preceding demyelination and axonal degeneration in CMT1C patients.

Description: Increased levels of nicotinamide/nicotinic acid mononucleotide adenylyltransferase ( NMNAT ) act as a powerful suppressor of Wallerian degeneration and ataxin- and tau-induced neurodegeneration in flies and mice. However, the nature of the suppression mechanism/s remains controversial. Here, we show that in yeast models of proteinopathies, overexpression of the NMNAT yeast homologs, NMA1 and NMA2 , suppresses polyglutamine (PolyQ) and α-synuclein-induced cytotoxicities. Unexpectedly, overexpression of other genes in the salvage pathway for NAD + biosynthesis, including QNS1 , NPT1 and PNC1 also protected against proteotoxicity. Our data revealed that in all cases, this mechanism involves extensive clearance of the non-native protein. Importantly, we demonstrate that suppression by NMA1 does not require the presence of a functional salvage pathway for NAD + biosynthesis, SIR2 or an active mitochondrial oxidative phosphorylation (OXPHOS) system. Our results imply the existence of histone deacetylase- and OXPHOS-independent crosstalk between the proteins in the salvage pathway for NAD + biosynthesis and the proteasome that can be manipulated to achieve cellular protection against proteotoxic stress.

Description: Defects in axonal transport are thought to contribute to the pathogenesis of neurodegenerative disease. Because α-tubulin acetylation facilitates axonal transport, inhibition of the α-tubulin deacetylating enzymes, histone deacetylase 6 (Hdac6) and silent information regulator 2 (Sirt2), is thought to be an interesting therapeutic strategy for these conditions. Amyotrophic lateral sclerosis (ALS) is a one such rapidly progressive and fatal neurodegenerative disorder, in which axonal transport defects have been found in vitro and in vivo . To establish whether the inhibition of Hdac6 or Sirt2 may be of interest for ALS treatment, we investigated whether deleting Hdac6 or Sirt2 from the superoxide dismutase 1, SOD1 G93A mouse affects the motor neuron degeneration in this ALS model. Deletion of Hdac6 significantly extended the survival of SOD1 G93A mice without affecting disease onset, and maintained motor axon integrity. This protective effect was associated with increased α-tubulin acetylation. Deletion of Sirt2 failed to affect the disease course, but also did not modify α-tubulin acetylation. These findings show that Hdac6, rather than Sirt2, is a therapeutic target for the treatment of ALS. Moreover, Sirt2 appears not to be a major α-tubulin deacetylase in the nervous system.

Description: The IL12B gene encodes the common p40 subunit of IL-12 and IL-23, cytokines with key roles in Th1 and Th17 biology, respectively, and genetic variation in this region significantly influences risk of psoriasis. Here, we demonstrate that a psoriasis-associated risk haplotype at the IL12B locus leads to increased expression of IL12B by monocytes and correlated with increased serum levels of IL-12, IFN- and the IFN- induced chemokine, CXCL10. In contrast, serum IL-23 levels were decreased in risk carriers when compared with non-carriers. We further demonstrate that IL-12 is increased in psoriatic skin and that risk carriers manifest a skewing of the inflammatory network toward stronger IFN- responses. Taken together, our data demonstrate that the risk variant in IL12B associates with its increased expression and predisposes to stronger Th1 polarization through deviation of the local inflammatory environment toward increased IL-12/IFN- at the expense of IL-23/IL-17 responses.

Description: Tc1 mouse model of Down syndrome (DS) is functionally trisomic for ~120 human chromosome 21 (HSA21) classical protein-coding genes. Tc1 mice display features relevant to the DS phenotype, including abnormalities in learning and memory and synaptic plasticity. To determine the molecular basis for the phenotypic features, the levels of 90 phosphorylation-specific and phosphorylation-independent proteins were measured by Reverse Phase Protein Arrays in hippocampus and cortex, and 64 in cerebellum, of Tc1 mice and littermate controls. Abnormal levels of proteins involved in MAP kinase, mTOR, GSK3B and neuregulin signaling were identified in trisomic mice. In addition, altered correlations among the levels of N-methyl-D-aspartate (NMDA) receptor subunits and the HSA21 proteins amyloid beta (A4) precursor protein (APP) and TIAM1, and between immediate early gene (IEG) proteins and the HSA21 protein superoxide dismutase-1 (SOD1) were found in the hippocampus of Tc1 mice, suggesting altered stoichiometry among these sets of functionally interacting proteins. Protein abnormalities in Tc1 mice were compared with the results of a similar analysis of Ts65Dn mice, a DS mouse model that is trisomic for orthologs of 50 genes trisomic in the Tc1 plus an additional 38 HSA21 orthologs. While there are similarities, abnormalities unique to the Tc1 include increased levels of the S100B calcium-binding protein, mTOR proteins RAPTOR and P70S6, the AMP-kinase catalytic subunit AMPKA, the IEG proteins FBJ murine osteosarcoma viral oncogene homolog (CFOS) and activity-regulated cytoskeleton-associated protein (ARC), and the neuregulin 1 receptor ERBB4. These data identify novel perturbations, relevant to neurological function and to some seen in Alzheimer's disease, that may occur in the DS brain, potentially contributing to phenotypic features and influencing drug responses.

Description: DNA methylation plays an important role in suppressing retrotransposon activity in mammalian genomes, yet there are stages of mammalian development where global hypomethylation puts the genome at risk of retrotransposition-mediated genetic instability. Hypomethylated primordial germ cells appear to limit this risk by expressing a cohort of retrotransposon-suppressing genome-defence genes whose silencing depends on promoter DNA methylation. Here, we investigate whether similar mechanisms operate in hypomethylated trophectoderm-derived components of the mammalian placenta to couple expression of genome-defence genes to the potential for retrotransposon activity. We show that the hypomethylated state of the mouse placenta results in activation of only one of the hypomethylation-sensitive germline genome-defence genes: Tex19.1. Tex19.1 appears to play an important role in placenta function as Tex19.1 –/– mouse embryos exhibit intra-uterine growth retardation and have small placentas due to a reduction in the number of spongiotrophoblast, glycogen trophoblast and sinusoidal trophoblast giant cells. Furthermore, we show that retrotransposon mRNAs are derepressed in Tex19.1 –/– placentas and that protein encoded by the LINE-1 retrotransposon is upregulated in hypomethylated trophectoderm-derived cells that normally express Tex19.1 . This study suggests that post-transcriptional genome-defence mechanisms are operating in the placenta to protect the hypomethylated cells in this tissue from retrotransposons and suggests that imbalances between retrotransposon activity and genome-defence mechanisms could contribute to placenta dysfunction and disease.

Description: Spinal Muscular Atrophy (SMA) is due to the loss of the survival motor neuron gene 1 (SMN1), resulting in motor neuron (MN) degeneration, muscle atrophy and loss of motor function. While SMN2 encodes a protein identical to SMN1, a single nucleotide difference in exon 7 causes most of the SMN2-derived transcripts to be alternatively spliced resulting in a truncated and unstable protein (SMN7). SMA patients retain at least one SMN2 copy, making it an important target for therapeutics. Many of the existing SMA models are very severe, with animals typically living less than 2 weeks. Here, we present a novel intermediate mouse model of SMA based upon the human genomic SMN2 gene. Genetically, this model is similar to the well-characterized SMN7 model; however, we have manipulated the SMN7 transgene to encode a modestly more functional protein referred to as SMN read-through (SMN RT ). By introducing the SMN RT transgene onto the background of a severe mouse model of SMA (SMN2 +/+ ;Smn –/– ), disease severity was significantly decreased based upon a battery of phenotypic parameters, including MN pathology and a significant extension in survival. Importantly, there is not a full phenotypic correction, allowing for the examination of a broad range of therapeutics, including SMN2-dependent and SMN-independent pathways. This novel animal model serves as an important biological and therapeutic model for less severe forms of SMA and provides an in vivo validation of the SMN RT protein.

Description: Manipulation of the mouse genome by site-specific mutagenesis has been extensively used to study gene function and model human disorders. Mouse models of myotubular myopathy (XLMTM), a severe congenital muscular disorder due to loss-of-function mutations in the MTM1 gene, have been generated by homologous recombination and shown that myotubularin is essential for skeletal muscle. However, since the Mtm1 deletion occurred constitutively or shortly after birth in these mice, it is not known whether myotubularin is required during adulthood, an important issue in the context of not only muscle biology but also therapies. To delete the Mtm1 gene in adult muscle fibers, we constructed a recombinant adeno-associated vector (AAV) that expresses the Cre recombinase under the muscle-specific desmin promoter. We report that a single injection of this vector into muscles of 3-month-old Mtm1 conditional mice leads to a myotubular myopathy phenotype with myofiber atrophy, disorganization of organelle positioning, such as mitochondria and nuclei, T-tubule defects and severe muscle weakness. In addition, our results show that MTM1-related atrophy and dysfunction correlate with abnormalities in satellite cell number and markers of autophagy, protein synthesis and neuromuscular junction transmission. The expression level of atrogenes was also analyzed. Therefore, we provide a valuable tissue model that recapitulates the main features of the disease, and it is useful to study pathogenesis and evaluate therapeutic strategies. We establish the proof-of-concept that myotubularin is required for the proper function of skeletal muscle during adulthood, suggesting that therapies will be required for the entire life of XLMTM patients.

Description: Polycystin 2 (Pkd2), which belongs to the transient receptor potential family, plays a critical role in development. Pkd2 is mainly localized in the primary cilia, which also function as mechanoreceptors in many cells that influence multiple biological processes including Ca 2+ influx, chemical activity and signalling pathways. Mutations in many cilia proteins result in craniofacial abnormalities. Orofacial tissues constantly receive mechanical forces and are known to develop and grow through intricate signalling pathways. Here we investigate the role of Pkd2, whose role remains unclear in craniofacial development and growth. In order to determine the role of Pkd2 in craniofacial development, we located expression in craniofacial tissues and analysed mice with conditional deletion of Pkd2 in neural crest-derived cells, using Wnt1Cre mice. Pkd2 mutants showed many signs of mechanical trauma such as fractured molar roots, distorted incisors, alveolar bone loss and compressed temporomandibular joints, in addition to abnormal skull shapes. Significantly, mutants showed no indication of any of these phenotypes at embryonic stages when heads perceive no significant mechanical stress in utero . The results suggest that Pkd2 is likely to play a critical role in craniofacial growth as a mechanoreceptor. Pkd2 is also identified as one of the genes responsible for autosomal dominant polycystic kidney disease (ADPKD). Since facial anomalies have never been identified in ADPKD patients, we carried out three-dimensional photography of patient faces and analysed these using dense surface modelling. This analysis revealed specific characteristics of ADPKD patient faces, some of which correlated with those of the mutant mice.

Description: WW domain-containing oxidoreductase (WWOX) is a tumor suppressor that has been reported to lose function due to genetic alterations in several cancers. WWOX maps to the common chromosomal fragile site FRA16D and several copy number variations (CNVs) were found within this gene. In this study, we investigated the association between the CNVs of WWOX and lung cancer risk in four independent case–control studies, which are on 2942 lung cancer cases and 3074 cancer-free controls of southern, eastern and northern Chinese. A common CNV-67048 was genotyped by the Taqman real-time PCR, and its biological effect was accessed with protein expression and sequencing assays. We found that in comparison with the common 2-copy genotype, the carriers of loss variant genotypes (1-copy or 0-copy) had a significantly increased risk of lung cancer (adjusted OR = 1.39, 95% CI = 1.24–1.55, P = 9.01 x 10 –9 ) in a dose–response manner ( P trend = 1.12 x 10 –10 ), and the WWOX protein expressions in lung cancer tissues were significantly lower ( P = 0.036), accompanying a higher rate of exons absence ( P = 0.021) in subjects with loss genotypes of CNV-67048. Our data suggest that the loss genotypes of CNV-67048 in WWOX predispose their carriers to lung cancer; this might be related with altered WWOX gene expression and exons absence in them.

Description: Sandhoff disease (SD) is a lysosomal storage disorder caused by a lack of a functional β-subunit of the β-hexosaminidase A and B enzymes, leading to the accumulation of gangliosides in the central nervous system (CNS). The Hexb –/– mouse model of SD shows a progressive neurodegenerative phenotype similar to the human equivalent. Previous studies have revealed that Hexb –/– mice suffer from chronic neuroinflammation characterized by microglial activation and expansion. Tumor necrosis factor-α (TNFα), a key modulator of the CNS immune response in models of neurodegeneration, is a hallmark of this activation. In this study, we explore the role of TNFα in the development and progression of SD in mice, by creating a Hexb –/– Tnfα –/– double-knockout mouse. Our results revealed that the double-knockout mice have an ameliorated disease course, with an extended lifespan, enhanced sensorimotor coordination and improved neurological function. TNFα-deficient SD mice also show decreased levels of astrogliosis and reduced neuronal cell death, with no alterations in neuronal storage of gangliosides. Interestingly, temporal microglia activation appears similar between the Hexb –/– Tnfα –/– and SD mice. Evidence is provided for the TNFα activation of the JAK2/STAT3 pathway as a mechanism for astrocyte activation in the disease. Bone marrow transplantation experiments reveal that both CNS-derived and bone marrow-derived TNFα have a pathological effect in SD mouse models, with CNS-derived TNFα playing a larger role. This study reveals TNFα as a neurodegenerative cytokine mediating astrogliosis and neuronal cell death in SD and points to TNFα as a potential therapeutic target to attenuate neuropathogenesis.

Description: The cone photoreceptor cyclic nucleotide-gated (CNG) channel is essential for central and color vision and visual acuity. Mutations in the channel subunits CNGA3 and CNGB3 are associated with achromatopsia and cone dystrophy. We investigated the gene expression profiles in mouse retina with CNG channel deficiency using whole genome expression microarrays. As cones comprise only 2 to 3% of the total photoreceptor population in the wild-type mouse retina, the mouse lines with CNG channel deficiency on a cone-dominant background, i.e. Cnga3 –/– /Nrl –/– and Cngb3 –/– /Nrl –/– mice, were used in our study. Comparative data analysis revealed a total of 105 genes altered in Cnga3 –/– /Nrl –/– and 92 in Cngb3 –/– /Nrl –/– retinas, relative to Nrl –/– retinas, with 27 genes changed in both genotypes. The differentially expressed genes primarily encode proteins associated with cell signaling, cellular function maintenance and gene expression. Ingenuity pathway analysis (IPA) identified 26 and 9 canonical pathways in Cnga3 –/– /Nrl –/– and Cngb3 –/– /Nrl –/– retinas, respectively, with 6 pathways being shared. The shared pathways include phototransduction, cAMP/PKA-mediated signaling, endothelin signaling, and EIF2/endoplasmic reticulum (ER) stress, whereas the IL-1, CREB, and purine metabolism signaling were found to specifically associate with Cnga3 deficiency. Thus, CNG channel deficiency differentially regulates genes that affect cell processes such as phototransduction, cellular survival and gene expression, and such regulations play a crucial role(s) in the retinal adaptation to impaired cone phototransduction. Though lack of Cnga3 and Cngb3 shares many common pathways, deficiency of Cnga3 causes more significant alterations in gene expression. This work provides insights into how cones respond to impaired phototransduction at the gene expression levels.

Description: Coenzyme Q10 (CoQ10) is essential for the energy production of the cells and as an electron transporter in the mitochondrial respiratory chain. CoQ10 links the mitochondrial fatty acid β-oxidation to the respiratory chain by accepting electrons from electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO). Recently, it was shown that a group of patients with the riboflavin responsive form of multiple acyl-CoA dehydrogenation deficiency (RR-MADD) carrying inherited amino acid variations in ETF-QO also had secondary CoQ10 deficiency with beneficial effects of CoQ10 treatment, thus adding RR-MADD to an increasing number of diseases involving secondary CoQ10 deficiency. In this study, we show that moderately decreased CoQ10 levels in fibroblasts from six unrelated RR-MADD patients were associated with increased levels of mitochondrial reactive oxygen species (ROS). Treatment with CoQ10, but not with riboflavin, could normalize the CoQ10 level and decrease the level of ROS in the patient cells. Additionally, riboflavin-depleted control fibroblasts showed moderate CoQ10 deficiency, but not increased mitochondrial ROS, indicating that variant ETF-QO proteins and not CoQ10 deficiency are the causes of mitochondrial ROS production in the patient cells. Accordingly, the corresponding variant Rhodobacter sphaeroides ETF-QO proteins, when overexpressed in vitro , bind a CoQ10 pseudosubstrate, Q10Br, less tightly than the wild-type ETF-QO protein, suggesting that molecular oxygen can get access to the electrons in the misfolded ETF-QO protein, thereby generating superoxide and oxidative stress, which can be reversed by CoQ10 treatment.

Description: 2-chloro-2-fluoro-deoxy-9-D-arabinofuranosyladenine (Clofarabine), a purine nucleoside analog, is used in the treatment of hematologic malignancies and as induction therapy for stem cell transplantation. The discovery of pharmacogenomic markers associated with chemotherapeutic efficacy and toxicity would greatly benefit the utility of this drug. Our objective was to identify genetic and epigenetic variants associated with clofarabine toxicity using an unbiased, whole genome approach. To this end, we employed International HapMap lymphoblastoid cell lines (190 LCLs) of European (CEU) or African (YRI) ancestry with known genetic information to evaluate cellular sensitivity to clofarabine. We measured modified cytosine levels to ascertain the contribution of genetic and epigenetic factors influencing clofarabine-mediated cytotoxicity. Association studies revealed 182 single nucleotide polymorphisms (SNPs) and 143 modified cytosines associated with cytotoxicity in both populations at the threshold P ≤ 0.0001. Correlation between cytotoxicity and baseline gene expression revealed 234 genes at P ≤ 3.98 x 10 –6 . Six genes were implicated as: (i) their expression was directly correlated to cytotoxicity, (ii) they had a targeting SNP associated with cytotoxicity, and (iii) they had local modified cytosines associated with gene expression and cytotoxicity. We identified a set of three SNPs and three CpG sites targeting these six genes explaining 43.1% of the observed variation in phenotype. siRNA knockdown of the top three genes ( SETBP1, BAG3, KLHL6 ) in LCLs revealed altered susceptibility to clofarabine, confirming relevance. As clofarabine's toxicity profile includes acute kidney injury, we examined the effect of siRNA knockdown in HEK293 cells. siSETBP1 led to a significant change in HEK293 cell susceptibility to clofarabine.

Description: Systemic sclerosis (SSc) and systemic lupus erythematosus (SLE) are two archetypal systemic autoimmune diseases which have been shown to share multiple genetic susceptibility loci. In order to gain insight into the genetic basis of these diseases, we performed a pan-meta-analysis of two genome-wide association studies (GWASs) together with a replication stage including additional SSc and SLE cohorts. This increased the sample size to a total of 21 109 (6835 cases and 14 274 controls). We selected for replication 19 SNPs from the GWAS data. We were able to validate KIAA0319L ( P = 3.31 x 10 –11 , OR = 1.49) as novel susceptibility loci for SSc and SLE. Furthermore, we also determined that the previously described SLE susceptibility loci PXK ( P = 3.27 x 10 –11 , OR = 1.20) and JAZF1 ( P = 1.11 x 10 –8 , OR = 1.13) are shared with SSc. Supporting these new discoveries, we observed that KIAA0319L was overexpressed in peripheral blood cells of SSc and SLE patients compared with healthy controls. With these, we add three ( KIAA0319L , PXK and JAZF1 ) and one ( KIAA0319L ) new susceptibility loci for SSc and SLE, respectively, increasing significantly the knowledge of the genetic basis of autoimmunity.

Description: In humans, the concerted action of at least 13 different peroxisomal PEX proteins is needed for proper peroxisome biogenesis. Mutations in any of these PEX genes can lead to lethal neurometabolic disorders of the Zellweger syndrome spectrum (ZSS). Previously, we identified the W313G mutation located within the SH3 domain of the peroxisomal protein, PEX13. As this tryptophan residue is highly conserved in almost all known SH3 proteins, we investigated the pathogenic mechanism of the W313G mutation and its role in PEX13 interactions and functions in peroxisome biogenesis. Here, we report for the first time that human PEX13 interacts with itself in peroxisomes in living cells. We demonstrate that the import of PTS1 (peroxisomal targeting signal 1) proteins is specifically disrupted when homooligomerization of PEX13 is interrupted. Live cell FRET microscopy in living cells as well as co-immunoprecipitation experiments reveal that the highly conserved W313 residue is important for self-association of PEX13 but is not required for interaction with PEX14, a well-established interaction partner at the peroxisomal membrane. Experiments with truncated constructs indicate that although the W313G mutation resides in the C-terminal SH3 domain, the N-terminal half is necessary for peroxisomal localization, which in turn appears to be crucial for homooligomerization. Furthermore, rescue of homooligomerization in the W313G mutant cells through complementation with truncation constructs restores import of peroxisomal matrix proteins. Taken together, the thorough analyses of a ZSS patient mutation unraveled the general cell biological function of PEX13 and its mechanism in the import of peroxisomal matrix PTS1 proteins.

Description: Primary cilia are cellular appendages important for signal transduction and sensing the environment. Bardet–Biedl syndrome proteins form a complex that is important for several cytoskeleton-related processes such as ciliogenesis, cell migration and division. However, the mechanisms by which BBS proteins may regulate the cytoskeleton remain unclear. We discovered that Bbs4- and Bbs6 -deficient renal medullary cells display a characteristic behaviour comprising poor migration, adhesion and division with an inability to form lamellipodial and filopodial extensions. Moreover, fewer mutant cells were ciliated [48% ± 6 for wild-type (WT) cells versus 23% ± 7 for Bbs4 null cells; P 〈 0.0001] and their cilia were shorter (2.55 μm ± 0.41 for WT cells versus 2.16 μm ± 0.23 for Bbs4 null cells; P 〈 0.0001). While the microtubular cytoskeleton and cortical actin were intact, actin stress fibre formation was severely disrupted, forming abnormal apical stress fibre aggregates. Furthermore, we observed over-abundant focal adhesions (FAs) in Bbs4- , Bbs6- and Bbs8 -deficient cells. In view of these findings and the role of RhoA in regulation of actin filament polymerization, we showed that RhoA-GTP levels were highly upregulated in the absence of Bbs proteins. Upon treatment of Bbs4 -deficient cells with chemical inhibitors of RhoA, we were able to restore the cilia length and number as well as the integrity of the actin cytoskeleton. Together these findings indicate that Bbs proteins play a central role in the regulation of the actin cytoskeleton and control the cilia length through alteration of RhoA levels.

Description: Huntington's disease (HD) is a neurodegenerative disorder caused by an abnormal expansion of a CAG repeat encoding a polyglutamine tract in the huntingtin (Htt) protein. The mutation leads to neuronal death through mechanisms which are still unknown. One hypothesis is that mitochondrial defects may play a key role. In support of this, the activity of mitochondrial complex II (C-II) is preferentially reduced in the striatum of HD patients. Here, we studied C-II expression in different genetic models of HD expressing N-terminal fragments of mutant Htt (mHtt). Western blot analysis showed that the expression of the 30 kDa Iron–Sulfur (Ip) subunit of C-II was significantly reduced in the striatum of the R6/1 transgenic mice, while the levels of the FAD containing catalytic 70 kDa subunit (Fp) were not significantly changed. Blue native gel analysis showed that the assembly of C-II in mitochondria was altered early in N171-82Q transgenic mice. Early loco-regional reduction in C-II activity and Ip protein expression was also demonstrated in a rat model of HD using intrastriatal injection of lentiviral vectors encoding mHtt. Infection of the rat striatum with a lentiviral vector coding the C-II Ip or Fp subunits induced a significant overexpression of these proteins that led to significant neuroprotection of striatal neurons against mHtt neurotoxicity. These results obtained in vivo support the hypothesis that structural and functional alterations of C-II induced by mHtt may play a critical role in the degeneration of striatal neurons in HD and that mitochondrial-targeted therapies may be useful in its treatment.

Description: Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are neurodegenerative disorders that are characterized by cytoplasmic aggregates and nuclear clearance of TAR DNA-binding protein 43 (TDP-43). Studies in Drosophila , zebrafish and mouse demonstrate that the neuronal dysfunction of TDP-43 is causally related to disease formation. However, TDP-43 aggregates are also observed in glia and muscle cells, which are equally affected in ALS and FTLD; yet, it is unclear whether glia- or muscle-specific dysfunction of TDP-43 contributes to pathogenesis. Here, we show that similar to its human homologue, Drosophila TDP-43, Tar DNA-binding protein homologue (TBPH), is expressed in glia and muscle cells. Muscle-specific knockdown of TBPH causes age-related motor abnormalities, whereas muscle-specific gain of function leads to sarcoplasmic aggregates and nuclear TBPH depletion, which is accompanied by behavioural deficits and premature lethality. TBPH dysfunction in glia cells causes age-related motor deficits and premature lethality. In addition, both loss and gain of Drosophila TDP-43 alter mRNA expression levels of the glutamate transporters Excitatory amino acid transporter 1 (EAAT1) and EAAT2. Taken together, our results demonstrate that both loss and gain of TDP-43 function in muscle and glial cells can lead to cytological and behavioural phenotypes in Drosophila that also characterize ALS and FTLD and identify the glutamate transporters EAAT1/2 as potential direct targets of TDP-43 function. These findings suggest that together with neuronal pathology, glial- and muscle-specific TDP-43 dysfunction may directly contribute to the aetiology and progression of TDP-43-related ALS and FTLD.

Description: RD3 is a 23 kDa protein implicated in the stable expression of guanylate cyclase in photoreceptor cells. Truncation mutations are responsible for photoreceptor degeneration and severe early-onset vision loss in Leber congenital amaurosis 12 (LCA12) patients, the rd3 mouse and the rcd2 collie. To further investigate the role of RD3 in photoreceptors and explore gene therapy as a potential treatment for LCA12, we delivered adeno-associated viral vector (AAV8) with a Y733F capsid mutation and containing the mouse Rd3 complementary DNA (cDNA) under the control of the human rhodopsin kinase promoter to photoreceptors of 14-day-old Rb(11.13)4Bnr/J and In (5)30Rk/J strains of rd3 mice by subretinal injections. Strong RD3 transgene expression led to the translocation of guanylate cyclase from the endoplasmic reticulum (ER) to rod and cone outer segments (OSs) as visualized by immunofluorescence microscopy. Guanylate cyclase expression and localization coincided with the survival of rod and cone photoreceptors for at least 7 months. Rod and cone visual function was restored in the In (5)30Rk/J strain of rd3 mice as measured by electroretinography (ERG), but only rod function was recovered in the Rb(11.13)4Bnr/J strain, suggesting that the latter may have another defect in cone phototransduction. These studies indicate that RD3 plays an essential role in the exit of guanylate cyclase from the ER and its trafficking to photoreceptor OSs and provide a ‘proof of concept’ for AAV-mediated gene therapy as a potential therapeutic treatment for LCA12.

Description: Bioenergetic dysfunction occurs in Alzheimer's disease (AD) and mild cognitive impairment (MCI), a clinical syndrome that frequently precedes symptomatic AD. In this study, we modeled AD and MCI bioenergetic dysfunction by transferring mitochondria from MCI, AD and control subject platelets to mtDNA-depleted SH-SY5Y cells. Bioenergetic fluxes and bioenergetics-related infrastructures were characterized in the resulting cytoplasmic hybrid (cybrid) cell lines. Relative to control cybrids, AD and MCI cybrids showed changes in oxygen consumption, respiratory coupling and glucose utilization. AD and MCI cybrids had higher ADP/ATP and lower NAD + /NADH ratios. AD and MCI cybrids exhibited differences in proteins that monitor, respond to or regulate cell bioenergetic fluxes including HIF1α, PGC1α, SIRT1, AMPK, p38 MAPK and mTOR. Several endpoints suggested mitochondrial mass increased in the AD cybrid group and probably to a lesser extent in the MCI cybrid group, and that the mitochondrial fission–fusion balance shifted towards increased fission in the AD and MCI cybrids. As many of the changes we observed in AD and MCI cybrid models are also seen in AD subject brains, we conclude reduced bioenergetic function is present during very early AD, is not brain-limited and induces protean retrograde responses that likely have both adaptive and mal-adaptive consequences.

Description: Fragile X Syndrome is the most common inherited cause of autism. Fragile X mental retardation protein (FMRP), which is absent in fragile X, is an mRNA binding protein that regulates the translation of hundreds of different mRNA transcripts. In the adult brain, FMRP is expressed primarily in the neurons; however, it is also expressed in developing glial cells, where its function is not well understood. Here, we show that fragile X (Fmr1) knockout mice display abnormalities in the myelination of cerebellar axons as early as the first postnatal week, corresponding roughly to the equivalent time in human brain development when symptoms of the syndrome first become apparent (1–3 years of age). At postnatal day (PND) 7, diffusion tensor magnetic resonance imaging showed reduced volume of the Fmr1 cerebellum compared with wild-type mice, concomitant with an 80–85% reduction in the expression of myelin basic protein, fewer myelinated axons and reduced thickness of myelin sheaths, as measured by electron microscopy. Both the expression of the proteoglycan NG2 and the number of PDGFRα+/NG2+ oligodendrocyte precursor cells were reduced in the Fmr1 cerebellum at PND 7. Although myelin proteins were still depressed at PND 15, they regained wild-type levels by PND 30. These findings suggest that impaired maturation or function of oligodendrocyte precursor cells induces delayed myelination in the Fmr1 mouse brain. Our results bolster an emerging recognition that white matter abnormalities in early postnatal brain development represent an underlying neurological deficit in Fragile X syndrome.

Description: More than 200 mutations in the skeletal muscle α-actin gene ( ACTA1 ) cause either dominant or recessive skeletal muscle disease. Currently, there are no specific therapies. Cardiac α-actin is 99% identical to skeletal muscle α-actin and the predominant actin isoform in fetal muscle. We previously showed cardiac α-actin can substitute for skeletal muscle α-actin, preventing the early postnatal death of Acta1 knock-out mice, which model recessive ACTA1 disease. Dominant ACTA1 disease is caused by the presence of ‘poison’ mutant actin protein. Experimental and anecdotal evidence nevertheless indicates that the severity of dominant ACTA1 disease is modulated by the relative amount of mutant skeletal muscle α-actin protein present. Thus, we investigated whether transgenic over-expression of cardiac α-actin in postnatal skeletal muscle could ameliorate the phenotype of mouse models of severe dominant ACTA1 disease. In one model, lethality of ACTA1 D286G . Acta1 +/– mice was reduced from ~59% before 30 days of age to ~12%. In the other model, Acta1 H40Y , in which ~80% of male mice die by 5 months of age, the cardiac α-actin transgene did not significantly improve survival. Hence cardiac α-actin over-expression is likely to be therapeutic for at least some dominant ACTA1 mutations. The reason cardiac α-actin was not effective in the Acta1 H40Y mice is uncertain. We showed that the Acta1 H40Y mice had endogenously elevated levels of cardiac α-actin in skeletal muscles, a finding not reported in dominant ACTA1 patients.

Description: Mutant superoxide dismutase 1 (SOD1) selectively associates with spinal cord mitochondria in rodent models of SOD1-mediated amyotrophic lateral sclerosis. A portion of mutant SOD1 exists in a non-native/misfolded conformation that is selectively recognized by conformational antibodies. Misfolded SOD1 is common to all mutant SOD1 models, is uniquely found in areas affected by the disease and is considered to mediate toxicity. We report that misfolded SOD1 recognized by the antibody B8H10 is present in greater abundance in mitochondrial fractions of SOD1 G93A rat spinal cords compared with oxidized SOD1, as recognized by the C4F6 antibody. Using a novel flow cytometric assay, we detect an age-dependent deposition of B8H10-reactive SOD1 on spinal cord mitochondria from both SOD1 G93A rats and SOD1 G37R mice. Mitochondrial damage, including increased mitochondrial volume, excess superoxide production and increased exposure of the toxic BH3 domain of Bcl-2, tracks positively with the presence of misfolded SOD1. Lastly, B8H10 reactive misfolded SOD1 is present in the lysates and mitochondrial fractions of lymphoblasts derived from ALS patients carrying SOD1 mutations, but not in controls. Together, these results highlight misfolded SOD1 as common to two ALS rodent animal models and familial ALS patient lymphoblasts with four different SOD1 mutations. Studies in the animal models point to a role for misfolded SOD1 in mitochondrial dysfunction in ALS pathogenesis.

Description: Genetic variation affecting absorption, distribution or excretion of essential trace elements may lead to health effects related to sub-clinical deficiency. We have tested for allelic effects of single-nucleotide polymorphisms (SNPs) on blood copper, selenium and zinc in a genome-wide association study using two adult cohorts from Australia and the UK. Participants were recruited in Australia from twins and their families and in the UK from pregnant women. We measured erythrocyte Cu, Se and Zn (Australian samples) or whole blood Se (UK samples) using inductively coupled plasma mass spectrometry. Genotyping was performed with Illumina chips and 〉2.5 m SNPs were imputed from HapMap data. Genome-wide significant associations were found for each element. For Cu, there were two loci on chromosome 1 (most significant SNPs rs1175550, P = 5.03 x 10 –10 , and rs2769264, P = 2.63 x 10 –20 ); for Se, a locus on chromosome 5 was significant in both cohorts (combined P = 9.40 x 10 –28 at rs921943); and for Zn three loci on chromosomes 8, 15 and X showed significant results (rs1532423, P = 6.40 x 10 –12 ; rs2120019, P = 1.55 x 10 –18 ; and rs4826508, P = 1.40 x 10 –12 , respectively). The Se locus covers three genes involved in metabolism of sulphur-containing amino acids and potentially of the analogous Se compounds; the chromosome 8 locus for Zn contains multiple genes for the Zn-containing enzyme carbonic anhydrase. Where potentially relevant genes were identified, they relate to metabolism of the element (Se) or to the presence at high concentration of a metal-containing protein (Cu).

Description: Heritable mutations in the BRCA1 and BRCA2 and other genes in the DNA double-strand break (DSB) repair pathway disrupt binding of the encoded proteins, transport into the nucleus and initiation of homologous recombination, thereby increasing cancer risk [Scully, R., Chen, J., Plug, A., Xiao, Y., Weaver, D., Feunteun, J., Ashley, T. and Livingston, D.M. (1997) Association of BRCA1 with Rad51 in mitotic and meiotic cells. Cell , 88, 265–275, Chen, J., Silver, D.P., Walpita, D., Cantor, S.B., Gazdar, A.F., Tomlinson, G., Couch, F.J., Weber, B.L., Ashley, T., Livingston, D.M. et al . (1998) Stable interaction between the products of the BRCA1 and BRCA2 tumor suppressor genes in mitotic and meiotic cells. Mol. Cell , 2, 317–328]. To meet the challenge of correct classification, flow cytometry-based functional variant analyses (FVAs) were developed to determine whether variants in DSB repair genes disrupted the binding of BRCA1 to BARD1, PALB2, BRCA2 and FANCD2, phosphorylation of p53 or BRCA1 nuclear localization in response to DNA damage caused by diepoxybutane, mitomycin C and bleomycin. Lymphoblastoid cells from individuals with BRCA1 pathogenic mutations, benign variants, and variants of uncertain significance or with known BRCA2 , FANCC or NBN mutations were tested. Mutations in BRCA1 decreased nuclear localization of BRCA1 in response to individual or combination drug treatment. Mutations in BRCA1 reduced binding to co-factors, PALB2 and FANCD2 and decreased phosphorylation of p53. Mutations in BRCA2 , FANCC and NBN decreased nuclear localization of BRCA1 in response to drug treatment, cofactors binding and p53 phosphorylation. Unsupervised cluster analysis of all and as few as two assays demonstrated two apparent clusters, high-risk BRCA1 mutations and phenocopies and low-risk, fully sequenced controls and variants of uncertain significance (VUS). Thus, two FVA assays distinguish BRCA1 mutations and phenocopies from benign variants and categorize most VUS as benign. Mutations in other DSB repair pathway genes produce molecular phenocopies. FVA assays may represent an adjunct to sequencing for categorizing VUS or may represent a stand-alone measure for assessing breast cancer risk.

Description: We analysed gene expression microarray data from whole blood samples from 228 multiple sclerosis (MS) patients either untreated or treated with one of three alternative commonly used interferon beta (IFNβ) disease modifying drugs: Avonex ® ( x 1 weekly), Betaseron ® (every second day) or Rebif ® ( x 3 weekly). Patient injections were not timed to coordinate sample collections, thus providing a global transcriptomic profile for each population of patients studied. Three hundred and fifty one genes were significantly differentially expressed by at least one of the IFNβ drugs. Despite the different drug sources with distinct injection and dosage protocols, a striking similarity was found in the identity and functional classes of the differentially expressed genes induced. Using the 25 most-upregulated genes, we defined a robust IFNβ gene expression signature that quantifies the IFN activation state per blood sample collected irrespective of the type of IFNβ therapy. This 25-gene signature also defined basal IFN activation states among untreated MS patients, which differed among individuals but remained relatively constant per patient with time. The maximum drug-induced IFN-activation state was similar for all three drugs despite a 1.7–2.0-fold diminished average effect for Avonex. This and a more erratic effect of Avonex per patient across longitudinal measurements is likely a result of its reduced injection frequency. In summary, we have defined a robust blood-derived type I IFN gene signature from MS patients. This signature could potentially serve to generically quantify the systemic Type I IFN activation status for any other clinical manifestation, inclusive of other autoimmune diseases.

Description: Acyl-CoA dehydrogenase 9 (ACAD9) is an assembly factor for mitochondrial respiratory chain Complex I (CI), and ACAD9 mutations are recognized as a frequent cause of CI deficiency. ACAD9 also retains enzyme ACAD activity for long-chain fatty acids in vitro , but the biological relevance of this function remains controversial partly because of the tissue specificity of ACAD9 expression: high in liver and neurons and minimal in skin fibroblasts. In this study, we hypothesized that this enzymatic ACAD activity is required for full fatty acid oxidation capacity in cells expressing high levels of ACAD9 and that loss of this function is important in determining phenotype in ACAD9-deficient patients. First, we confirmed that HEK293 cells express ACAD9 abundantly. Then, we showed that ACAD9 knockout in HEK293 cells affected long-chain fatty acid oxidation along with Cl, both of which were rescued by wild type ACAD9 . Further, we evaluated whether the loss of ACAD9 enzymatic fatty acid oxidation affects clinical severity in patients with ACAD9 mutations. The effects on ACAD activity of 16 ACAD9 mutations identified in 24 patients were evaluated using a prokaryotic expression system. We showed that there was a significant inverse correlation between residual enzyme ACAD activity and phenotypic severity of ACAD9-deficient patients. These results provide evidence that in cells where it is strongly expressed, ACAD9 plays a physiological role in fatty acid oxidation, which contributes to the severity of the phenotype in ACAD9-deficient patients. Accordingly, treatment of ACAD9 patients should aim at counteracting both CI and fatty acid oxidation dysfunctions.

Description: Mutations in the ABCA4 gene are a common cause of autosomal recessive retinal degeneration. All mouse models to date are based on knockouts of Abca4 , even though the disease is often caused by missense mutations such as the complex allele L541P;A1038V (PV). We now show that the PV mutation causes severe human disease whereas the V mutation alone causes mild disease. Mutant ABCA4 proteins expressed heterologously in mammalian cells retained normal cellular localization. However, basal and all- trans -retinal-stimulated ATPase activities were reduced substantially for P and PV but only mildly for V. Electron microscopy revealed marked structural changes and misfolding for the P and PV mutants but few changes for the V mutant, consistent with the disease severity difference in patients. We generated Abca4 PV/PV knock-in mice homozygous for the complex PV allele to investigate the effects of this misfolding mutation in vivo . Mutant ABCA4 RNA levels approximated WT ABCA4 RNA levels but, surprisingly, only trace amounts of mutant ABCA4 protein were noted in the retina. RNA sequencing of WT, Abca4 –/– and Abca4 PV/PV mice revealed mild gene expression alterations in the retina and RPE. Similar to Abca4 –/– mice, Abca4 PV/PV mice showed substantial A2E and lipofuscin accumulation in their RPE cells but no retinal degeneration up to 12 months of age. Thus, rapid degradation of this large misfolded mutant protein in mouse retina caused little detectable photoreceptor degeneration. These findings suggest likely differences in the unfolded protein response between murine and human photoreceptors and support development of therapies directed at increasing this capability in patients.

Description: PLEC , the gene encoding the cytolinker protein plectin, has eight tissue-specific isoforms in humans, arising by alternate splicing of the first exon. To date, all PLEC mutations that cause epidermolysis bullosa simplex (EBS) were found in exons common to all isoforms. Due to the ubiquitous presence of plectin in mammalian tissues, EBS from recessive plectin mutations is always associated with extracutaneous involvement including muscular dystrophy, pyloric atresia and cardiomyopathy. We studied a consanguineous family with sisters having isolated blistering suggesting EBS. Skin disease started with foot blisters at walking age and became generalized at puberty while sparing mucous membranes. DNA sequencing revealed a homozygous nonsense mutation (c.46C〉T; p.Arg16X) in the first exon of the plectin variant encoding plectin isoform 1a (P1a). Immunofluorescence antigen mapping, transmission electron microscopy, western blot analysis and qRT-PCR were performed on patient skin and cultured keratinocytes, control myocardium and striated muscle samples. We found hypoplastic hemidesmosomes and intra-epidermal ‘pseudo-junctional’ cleavage fitting EBS. Screening for cardiomyopathy and muscle dystrophy showed no abnormalities. We report the first cases of autosomal-recessive EBS from P1a deficiency affecting skin, while mucous membranes, heart and muscle are spared. The dominant expression of the P1a isoform in epidermal basal cell layer and cultured keratinocytes suggests that mutations in the first exon of isoform 1a cause skin-only EBS without extracutaneous involvement. Our study characterizes yet another of the eight isoforms of plectin and adds a tissue-specific phenotype to the spectrum of ‘plectinopathies' produced by mutations of unique first exons of this gene.

Description: Carotenoids are currently investigated regarding their potential to lower the risk of chronic disease and to combat vitamin A deficiency. Surprisingly, responses to dietary supplementation with these compounds are quite variable between individuals. Genome-wide studies have associated common genetic polymorphisms in the BCO1 gene with this variability. The BCO1 gene encodes an enzyme that is expressed in the intestine and converts provitamin A carotenoids to vitamin A-aldehyde. However, it is not clear how this enzyme can impact the bioavailability and metabolism of other carotenoids such as xanthophyll. We here provide evidence that BCO1 is a key component of a regulatory network that controls the absorption of carotenoids and fat-soluble vitamins. In this process, conversion of β-carotene to vitamin A by BCO1 induces via retinoid signaling the expression of the intestinal homeobox transcription factor ISX. Subsequently, ISX binds to conserved DNA-binding motifs upstream of the BCO1 and SCARB1 genes. SCARB1 encodes a membrane protein that facilitates absorption of fat-soluble vitamins and carotenoids. In keeping with its role as a transcriptional repressor, SCARB1 protein levels are significantly increased in the intestine of ISX-deficient mice. This increase results in augmented absorption and tissue accumulation of xanthophyll carotenoids and tocopherols. Our study shows that fat-soluble vitamin and carotenoid absorption is controlled by a BCO1-dependent negative feedback regulation. Thus, our findings provide a molecular framework for the controversial relationship between genetics and fat-soluble vitamin status in the human population.

Description: Huntington's disease (HD) is a fatal neurodegenerative disease, caused by expansion of polyglutamine repeats in the Huntingtin gene, with longer expansions leading to earlier ages of onset. The HD iPSC Consortium has recently reported a new in vitro model of HD based on the generation of induced pluripotent stem cells (iPSCs) from HD patients and controls. The current study has furthered the disease in a dish model of HD by generating new non-integrating HD and control iPSC lines. Both HD and control iPSC lines can be efficiently differentiated into neurons/glia; however, the HD-derived cells maintained a significantly greater number of nestin-expressing neural progenitor cells compared with control cells. This cell population showed enhanced vulnerability to brain-derived neurotrophic factor (BDNF) withdrawal in the juvenile-onset HD (JHD) lines, which appeared to be CAG repeat-dependent and mediated by the loss of signaling from the TrkB receptor. It was postulated that this increased death following BDNF withdrawal may be due to glutamate toxicity, as the N -methyl- d -aspartate (NMDA) receptor subunit NR2B was up-regulated in the cultures. Indeed, blocking glutamate signaling, not just through the NMDA but also mGlu and AMPA/Kainate receptors, completely reversed the cell death phenotype. This study suggests that the pathogenesis of JHD may involve in part a population of ‘persistent’ neural progenitors that are selectively vulnerable to BDNF withdrawal. Similar results were seen in adult hippocampal-derived neural progenitors isolated from the BACHD model mouse. Together, these results provide important insight into HD mechanisms at early developmental time points, which may suggest novel approaches to HD therapeutics.

Description: Fabry disease is caused by deficient activity of lysosomal enzyme α-galactosidase A. The enzyme deficiency results in intracellular accumulation of glycosphingolipids, leading to a variety of clinical manifestations including hypertrophic cardiomyopathy and renal insufficiency. The mechanism through which glycosphingolipid accumulation causes these manifestations remains unclear. Current treatment, especially when initiated at later stage of the disease, does not produce completely satisfactory results. Elucidation of the pathogenesis of Fabry disease is therefore crucial to developing new treatments. We found increased activity of androgen receptor (AR) signaling in Fabry disease. We subsequently also found that blockade of AR signaling either through castration or AR-antagonist prevented and reversed cardiac and kidney hypertrophic phenotype in a mouse model of Fabry disease. Our findings implicate abnormal AR pathway in the pathogenesis of Fabry disease and suggest blocking AR signaling as a novel therapeutic approach.

Description: We describe the case of a woman in whom combination of a mitochondrial ( MT-CYB ) and a nuclear ( SDHB ) mutation was associated with clinical and metabolic features suggestive of a mitochondrial disorder. The mutations impaired overall energy metabolism in the patient's muscle and fibroblasts and increased cellular susceptibility to oxidative stress. To clarify the contribution of each mutation to the phenotype, mutant yeast strains were generated. A significant defect in strains carrying the Sdh2 mutation, either alone or in combination with the cytb variant, was observed. Our data suggest that the SDHB mutation was causative of the mitochondrial disorder in our patient with a possible cumulative contribution of the MT-CYB variant. To our knowledge, this is the first association of bi-genomic variants in the mtDNA and in a nuclear gene encoding a subunit of complex II.

Description: Paget's disease of bone (PDB) is a common disorder with a strong genetic component characterized by increased but disorganized bone remodelling. Previous genome-wide association studies identified a locus on chromosome 14q32 tagged by rs10498635 which was significantly associated with susceptibility to PDB in several European populations. Here we conducted fine-mapping and targeted sequencing of the candidate locus to identify possible functional variants. Imputation in 741 PDB patients and 2699 controls confirmed that the association was confined to a 60 kb region in the RIN3 gene and conditional analysis adjusting for rs10498635 identified no new independent signals. Sequencing of the RIN3 gene identified a common missense variant (p.R279C) that was strongly associated with the disease (OR = 0.64; P = 1.4 x 10 –9 ), and was in strong linkage disequilibrium with rs10498635. A further 13 rare missense variants were identified, seven of which were novel and detected only in PDB cases. When combined, these rare variants were over-represented in cases compared with controls (OR = 3.72; P = 8.9 x 10 –10 ). Most rare variants were located in a region that encodes a proline-rich, intrinsically disordered domain of the protein and many were predicted to be pathogenic. RIN3 was expressed in bone tissue and its expression level was ~10-fold higher in osteoclasts compared with osteoblasts. We conclude that susceptibility to PDB at the 14q32 locus is mediated by a combination of common and rare coding variants in RIN3 and suggest that RIN3 may contribute to PDB susceptibility by affecting osteoclast function.

Description: PTEN-induced kinase 1 (Pink1) and ubiquitin E3 ligase Parkin function in a linear pathway to maintain healthy mitochondria via regulating mitochondrial clearance and trafficking. Mutations in the two enzymes cause the familial form of Parkinson's disease (PD) in humans, as well as accumulation of defective mitochondria and cellular degeneration in flies. Here, we show that loss of function of a scaffolding protein Mask, also known as ANKHD1 (Ankyrin repeats and KH domain containing protein 1) in humans, rescues the behavioral, anatomical and cellular defects caused by pink1 or parkin mutations in a cell-autonomous manner. Moreover, similar rescue can also be achieved if Mask knock-down is induced in parkin adult flies when the mitochondrial dystrophy is already manifested. We found that Mask genetically interacts with Parkin to modulate mitochondrial morphology and negatively regulates the recruitment of Parkin to mitochondria. We also provide evidence that loss of Mask activity promotes co-localization of the autophagosome marker with mitochondria in developing larval muscle, and that an intact autophagy pathway is required for the rescue of parkin mutant defects by mask loss of function. Together, our data strongly suggest that Mask/ANKHD1 activity can be inhibited in a tissue- and timely-controlled fashion to restore mitochondrial integrity under PD-linked pathological conditions.

Description: Actinic keratosis (AK) is a pre-malignant skin disease, highly prevalent in elderly Europeans. This study investigates genetic susceptibility to AK with a genome-wide association study (GWAS). A full body skin examination was performed in 3194 elderly individuals from the Rotterdam Study (RS) of exclusive north-western European origin (aged 51–99 years, 45% male). Physicians graded the number of AK into four severity levels: none (76%), 1–3 (14%), 4–9 (6%) and ≥10 (5%), and skin color was quantified using a spectrophotometer on sun-unexposed skin. A GWAS for AK severity was conducted, where promising signals at IRF4 and MC1R ( P 〈 4.2 x 10 –7 ) were successfully replicated in an additional cohort of 623 RS individuals ( IRF4 , rs12203592, P combined = 6.5 x 10 –13 and MC1R , rs139810560, P combined = 4.1 x 10 –9 ). Further, in an analysis of ten additional well-known human pigmentation genes, TYR also showed significant association with AK (rs1393350, P = 5.3 x 10 –4 ) after correction for multiple testing. Interestingly, the strength and significance of above-mentioned associations retained largely the same level after skin color adjustment. Overall, our data strongly suggest that IRF4 , MC1R and TYR genes likely have pleiotropic effects, a combination of pigmentation and oncogenic functions, resulting in an increased risk of AK.

Description: Recent advances in genetics have spurred rapid progress towards the systematic identification of genes involved in complex diseases. Still, the detailed understanding of the molecular and physiological mechanisms through which these genes affect disease phenotypes remains a major challenge. Here, we identify the asthma disease module , i.e. the local neighborhood of the interactome whose perturbation is associated with asthma, and validate it for functional and pathophysiological relevance, using both computational and experimental approaches. We find that the asthma disease module is enriched with modest GWAS P -values against the background of random variation, and with differentially expressed genes from normal and asthmatic fibroblast cells treated with an asthma-specific drug. The asthma module also contains immune response mechanisms that are shared with other immune-related disease modules. Further, using diverse omics (genomics, gene-expression, drug response) data, we identify the GAB1 signaling pathway as an important novel modulator in asthma. The wiring diagram of the uncovered asthma module suggests a relatively close link between GAB1 and glucocorticoids (GCs), which we experimentally validate, observing an increase in the level of GAB1 after GC treatment in BEAS-2B bronchial epithelial cells. The siRNA knockdown of GAB1 in the BEAS-2B cell line resulted in a decrease in the NFkB level, suggesting a novel regulatory path of the pro-inflammatory factor NFkB by GAB1 in asthma.

Description: Exposure of a developing foetus to maternal gestational diabetes (GDM) has been shown to programme future risk of diabetes and obesity. Epigenetic variation in foetal tissue may have a mechanistic role in metabolic disease programming through interaction of the pregnancy environment with gene function. We aimed to identify genome-wide DNA methylation variation in cord blood and placenta from offspring born to mothers with and without GDM. Pregnant women of South Asian origin were studied and foetal tissues sampled at term delivery. The Illumina HumanMethylation450 BeadChip was used to assay genome-wide DNA methylation in placenta and cord blood from 27 GDM exposed and 21 unexposed offspring. We identified 1485 cord blood and 1708 placenta methylation variable positions (MVPs) achieving genome-wide significance (adjusted P -value 〈0.05) with methylation differences of 〉5%. MVPs were disproportionately located within first exons. A bioinformatic co-methylation algorithm was used to detect consistent directionality of methylation in 1000 bp window around each MVP was observed at 74% of placenta and 59% of cord blood MVPs. KEGG pathway analysis showed enrichment of pathways involved in endocytosis, MAPK signalling and extracellular triggers to intracellular metabolic processes. Replication studies should integrate genomics and transcriptomics with longitudinal sampling to elucidate stability, determine causality for translation into biomarker and prevention studies.

Description: Inherited dental malformations constitute a clinically and genetically heterogeneous group of disorders. Here, we report on four families, three of them consanguineous, with an identical phenotype, characterized by significant short stature with brachyolmia and hypoplastic amelogenesis imperfecta (AI) with almost absent enamel. This phenotype was first described in 1996 by Verloes et al. as an autosomal recessive form of brachyolmia associated with AI. Whole-exome sequencing resulted in the identification of recessive hypomorphic mutations including deletion, nonsense and splice mutations, in the LTBP3 gene, which is involved in the TGF-beta signaling pathway. We further investigated gene expression during mouse development and tooth formation. Differentiated ameloblasts synthesizing enamel matrix proteins and odontoblasts expressed the gene. Study of an available knockout mouse model showed that the mutant mice displayed very thin to absent enamel in both incisors and molars, hereby recapitulating the AI phenotype in the human disorder.

Description: Disarrangement in functions and quality control of mitochondria at synapses are early events in Alzheimer's disease (AD) pathobiology. We reported that a 20–22 kDa NH 2 -tau fragment mapping between 26 and 230 amino acids of the longest human tau isoform (aka NH 2 htau): (i) is detectable in cellular and animal AD models, as well in synaptic mitochondria and cerebrospinal fluids (CSF) from human AD subjects; (ii) is neurotoxic in primary hippocampal neurons; (iii) compromises the mitochondrial biology both directly, by inhibiting the ANT-1-dependent ADP/ATP exchange, and indirectly, by impairing their selective autophagic clearance (mitophagy). Here, we show that the extensive Parkin-dependent turnover of mitochondria occurring in NH 2 htau-expressing post-mitotic neurons plays a pro-death role and that UCHL-1, the cytosolic Ubiquitin-C-terminal hydrolase L1 which directs the physiological remodeling of synapses by controlling ubiquitin homeostasis, critically contributes to mitochondrial and synaptic failure in this in vitro AD model. Pharmacological or genetic suppression of improper mitophagy, either by inhibition of mitochondrial targeting to autophagosomes or by shRNA-mediated silencing of Parkin or UCHL-1 gene expression, restores synaptic and mitochondrial content providing partial but significant protection against the NH 2 htau-induced neuronal death. Moreover, in mitochondria from human AD synapses, the endogenous NH 2 htau is stably associated with Parkin and with UCHL-1. Taken together, our studies show a causative link between the excessive mitochondrial turnover and the NH 2 htau-induced in vitro neuronal death, suggesting that pathogenetic tau truncation may contribute to synaptic deterioration in AD by aberrant recruitment of Parkin and UCHL-1 to mitochondria making them more prone to detrimental autophagic clearance.

Description: Temporal lobe epilepsy (TLE) is a common epilepsy syndrome with a complex etiology. Despite evidence for the participation of genetic factors, the genetic basis of TLE remains largely unknown. A role for the galanin neuropeptide in the regulation of epileptic seizures has been established in animal models more than two decades ago. However, until now there was no report of pathogenic mutations in GAL , the galanin-encoding gene, and therefore its role in human epilepsy was not established. Here, we studied a family with a pair of monozygotic twins affected by TLE and two unaffected siblings born to healthy parents. Exome sequencing revealed that both twins carried a novel de novo mutation (p.A39E) in the GAL gene. Functional analysis revealed that the p.A39E mutant showed antagonistic activity against galanin receptor 1 (GalR1)-mediated response, and decreased binding affinity and reduced agonist properties for GalR2. These findings suggest that the p.A39E mutant could impair galanin signaling in the hippocampus, leading to increased glutamatergic excitation and ultimately to TLE. In a cohort of 582 cases, we did not observe any pathogenic mutations indicating that mutations in GAL are a rare cause of TLE. The identification of a novel de novo mutation in a biologically-relevant candidate gene, coupled with functional evidence that the mutant protein disrupts galanin signaling, strongly supports GAL as the causal gene for the TLE in this family. Given the availability of galanin agonists which inhibit seizures, our findings could potentially have direct implications for the development of anti-epileptic treatment.

Description: Foxl2 codes for a forkhead/HNF3 transcription factor essential for follicular maturation and maintenance of ovarian identity. FOXL2 mutations are associated with Blepharophimosis, Ptosis and Epicanthus inversus Syndrome (BPES) characterized by eyelid malformations (types I and II) and premature ovarian insufficiency (type I). We show that Foxl2 is not only expressed by the ovary, but also by other components of the mouse female reproductive tract, including the uterus, the cervix and the oviduct. In the uterus, Foxl2 expression is first observed in the neonatal mesenchyme and, during uterine maturation, persists in the stroma and in the deep inner myometrial layer (IML). In the adult, Foxl2 is expressed in the differentiated stromal layer, but no longer in the myometrium. Conditional deletion of Foxl2 in the postnatal (PN) uterus using Progesterone Receptor-cre ( Pgr cre/+ ) mice results in infertility. During PN uterine maturation Pgr cre/+ ; Foxl2 flox/flox mice present a severely reduced thickness of the stroma layer and an hypertrophic, disorganized IML. In adult Pgr cre/+ ; Foxl2 flox/flox mice a supplementary muscular layer is present at the stroma/myometrium border and vascular smooth muscle cells fail to form a coherent layer around uterine arteries. Wnt signalling pathways play a central role in uterine maturation; in Pgr cre/+ ; Foxl2 flox/flox mice, Wnt genes are deregulated suggesting that Foxl2 acts through these signals. In humans, thickening of the IML (also called "junctional zone") is associated with reduced fertility, endometriosis and adenomyosis. Our data suggest that Foxl2 has a crucial role in PN uterine maturation and could help to understand sub-fertility predisposition in women.

Description: Mutations in the Crumbs-homologue-1 ( CRB1 ) gene lead to severe recessive inherited retinal dystrophies. Gene transfer therapy is the most promising cure for retinal dystrophies and has primarily been applied for recessive null conditions via a viral gene expression vector transferring a cDNA encoding an enzyme or channel protein, and targeting expression to one cell type. Therapy for the human CRB1 disease will be more complex, as CRB1 is a structural and signaling transmembrane protein present in three cell classes: Müller glia, cone and rod photoreceptors. In this study, we applied CRB1 and CRB2 gene therapy vectors in Crb1 -retinitis pigmentosa mouse models at mid-stage disease. We tested if CRB expression restricted to Müller glial cells or photoreceptors or co-expression in both is required to recover retinal function. We show that targeting both Müller glial cells and photoreceptors with CRB2 ameliorated retinal function and structure in Crb1 mouse models. Surprisingly, targeting a single cell type or all cell types with CRB1 reduced retinal function. We show here the first pre-clinical studies for CRB1 -related eye disorders using CRB2 vectors and initial elucidation of the cellular mechanisms underlying CRB1 function.

Description: Chordin-Like 1 ( CHRDL1 ) mutations cause non-syndromic X-linked megalocornea (XMC) characterized by enlarged anterior eye segments. Mosaic corneal degeneration, presenile cataract and secondary glaucoma are associated with XMC. Beside that CHRDL1 encodes Ventroptin, a secreted bone morphogenetic protein (BMP) antagonist, the molecular mechanism of XMC is not well understood yet. In a family with broad phenotypic variability of XMC, we identified the novel CHRDL1 frameshift mutation c.807_808delTC [p.H270Wfs*22] presumably causing CHRDL1 loss of function. Using Xenopus laevis as model organism, we demonstrate that chrdl1 is specifically expressed in the ocular tissue at late developmental stages. The chrdl1 knockdown directly resembles the human XMC phenotype and confirms CHRDL1 deficiency to cause XMC. Interestingly, secondary to this bmp4 is down-regulated in the Xenopus eyes. Moreover, phospho-SMAD1/5 is altered and BMP receptor 1A is reduced in a XMC patient. Together, we classify these observations as negative-feedback regulation due to the deficient BMP antagonism in XMC. As CHRDL1 is preferentially expressed in the limbal stem cell niche of adult human cornea, we assume that CHRDL1 plays a key role in cornea homeostasis. In conclusion, we provide novel insights into the molecular mechanism of XMC as well as into the specific role of CHRDL1 during cornea organogenesis, among others by the establishment of the first XMC in vivo model. We show that unravelling monogenic cornea disorders like XMC—with presumably disturbed cornea growth and differentiation—contribute to the identification of potential limbal stem cell niche factors that are promising targets for regenerative therapies of corneal injuries.

Description: A GGGGCC-repeat expansion in C9orf72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) among Caucasians. However, little is known about the variability of the GGGGCC expansion in different tissues and whether this correlates with the observed phenotype. Here, we used Southern blotting to estimate the size of hexanucleotide expansions in C9orf72 in neural and non-neural tissues from 18 autopsied ALS and FTD patients with repeat expansion in blood. Digitalization of the Southern blot images allowed comparison of repeat number, smear distribution and expansion band intensity between tissues and between patients. We found marked intra-individual variation of repeat number between tissues, whereas there was less variation within each tissue group. In two patients, the size variation between tissues was extreme, with repeat numbers below 100 in all studied non-neural tissues, whereas expansions in neural tissues were 20–40 times greater and in the same size range observed in neural tissues of the other 16 patients. The expansion pattern in different tissues could not distinguish between diagnostic groups and no correlation was found between expansion size in frontal lobe and occurrence of cognitive impairment. In ALS patients, a less number of repeats in the cerebellum and parietal lobe correlated with earlier age of onset and a larger number of repeats in the parietal lobe correlated with a more rapid progression. In 43 other individuals without repeat expansion in blood, we find that repeat sizes up to 15 are stable, as no size variation between blood, brain and spinal cord was found.

Description: Chromosomal rearrangements with duplication of the lamin B1 ( LMNB1 ) gene underlie autosomal dominant adult-onset demyelinating leukodystrophy (ADLD), a rare neurological disorder in which overexpression of LMNB1 causes progressive central nervous system demyelination. However, we previously reported an ADLD family (ADLD-1-TO) without evidence of duplication or other mutation in LMNB1 despite linkage to the LMNB1 locus and lamin B1 overexpression. By custom array-CGH, we further investigated this family and report here that patients carry a large (~660 kb) heterozygous deletion that begins 66 kb upstream of the LMNB1 promoter. Lamin B1 overexpression was confirmed in further ADLD-1-TO tissues and in a postmortem brain sample, where lamin B1 was increased in the frontal lobe. Through parallel studies, we investigated both loss of genetic material and chromosomal rearrangement as possible causes of LMNB1 overexpression, and found that ADLD-1-TO plausibly results from an enhancer adoption mechanism. The deletion eliminates a genome topological domain boundary, allowing normally forbidden interactions between at least three forebrain-directed enhancers and the LMNB1 promoter, in line with the observed mainly cerebral localization of lamin B1 overexpression and myelin degeneration. This second route to LMNB1 overexpression and ADLD is a new example of the relevance of regulatory landscape modifications in determining Mendelian phenotypes.

Description: In mitochondria, carbamoyl-phosphate synthetase 1 activity produces carbamoyl phosphate for urea synthesis, and deficiency results in hyperammonemia. Cytoplasmic carbamoyl-phosphate synthetase 2, however, is part of a tri-functional enzyme encoded by CAD ; no human disease has been attributed to this gene. The tri-functional enzyme contains carbamoyl-phosphate synthetase 2 (CPS2), aspartate transcarbamylase (ATCase) and dihydroorotase (DHOase) activities, which comprise the first three of six reactions required for de novo pyrimidine biosynthesis. Here we characterize an individual who is compound heterozygous for mutations in different domains of CAD. One mutation , c.1843-1G〉A, results in an in-frame deletion of exon 13. The other, c.6071G〉A, causes a missense mutation (p.Arg2024Gln) in a highly conserved residue that is essential for carbamoyl-phosphate binding. Metabolic flux studies showed impaired aspartate incorporation into RNA and DNA through the de novo synthesis pathway. In addition, CTP, UTP and nearly all UDP-activated sugars that serve as donors for glycosylation were decreased. Uridine supplementation rescued these abnormalities, suggesting a potential therapy for this new glycosylation disorder.

Description: mRNA decay is an essential and active process that allows cells to continuously adapt gene expression to internal and environmental cues. There are two mRNA degradation pathways: 3' to 5' and 5' to 3'. The DCPS protein is the scavenger mRNA decapping enzyme which functions in the last step of the 3' end mRNA decay pathway. We have identified a DCPS pathogenic mutation in a large family with three affected individuals presenting with a novel recessive syndrome consisting of craniofacial anomalies, intellectual disability and neuromuscular defects. Using patient's primary cells, we show that this homozygous splice mutation results in a DCPS loss-of-function allele. Diagnostic biochemical analyses using various m 7 G cap derivatives as substrates reveal no DCPS enzymatic activity in patient's cells. Our results implicate DCPS and more generally RNA catabolism, as a critical cellular process for neurological development, normal cognition and organismal homeostasis in humans.

Description: There are two known mRNA degradation pathways, 3' to 5' and 5' to 3'. We identified likely pathogenic variants in two genes involved in these two pathways in individuals with intellectual disability. In a large family with multiple branches, we identified biallelic variants in DCPS in three affected individuals; a splice site variant (c.636+1G〉A) that results in an in-frame insertion of 45 nucleotides and a missense variant (c.947C〉T; p.Thr316Met). DCPS decaps the cap structure generated by 3' to 5' exonucleolytic degradation of mRNA. In vitro decapping assays showed an ablation of decapping function for both variants in DCPS. In another family, we identified a homozygous mutation (c.161T〉C; p.Phe54Ser) in EDC3 in two affected children. EDC3 stimulates DCP2, which decaps mRNAs at the beginning of the 5' to 3' degradation pathway. In vitro decapping assays showed that altered EDC3 is unable to enhance DCP2 decapping at low concentrations and even inhibits DCP2 decapping at high concentration. We show that individuals with biallelic mutations in these genes of seemingly central functions are viable and that these possibly lead to impairment of neurological functions linking mRNA decapping to normal cognition. Our results further affirm an emerging theme linking aberrant mRNA metabolism to neurological defects.

Description: Clinical and neuropathological similarities between dementia with Lewy bodies (DLB), Parkinson's and Alzheimer's diseases (PD and AD, respectively) suggest that these disorders may share etiology. To test this hypothesis, we have performed an association study of 54 genomic regions, previously implicated in PD or AD, in a large cohort of DLB cases and controls. The cohort comprised 788 DLB cases and 2624 controls. To minimize the issue of potential misdiagnosis, we have also performed the analysis including only neuropathologically proven DLB cases (667 cases). The results show that the APOE is a strong genetic risk factor for DLB, confirming previous findings, and that the SNCA and SCARB2 loci are also associated after a study-wise Bonferroni correction, although these have a different association profile than the associations reported for the same loci in PD. We have previously shown that the p.N370S variant in GBA is associated with DLB, which, together with the findings at the SCARB2 locus, suggests a role for lysosomal dysfunction in this disease. These results indicate that DLB has a unique genetic risk profile when compared with the two most common neurodegenerative diseases and that the lysosome may play an important role in the etiology of this disorder. We make all these data available.

Description: Mitochondrial DNA mutations at MT-ATP6 gene are relatively common in individuals suffering from striatal necrosis syndromes. These patients usually do not show apparent histochemical and/or biochemical signs of oxidative phosphorylation dysfunction. Because of this, MT-ATP6 is not typically analyzed in many other mitochondrial disorders that have not been previously associated to mutations in this gene. To correct this bias, we have performed a screening of the MT-ATP6 gene in a large collection of patients suspected of suffering different mitochondrial DNA (mtDNA) disorders. In three cases, biochemical, molecular-genetics and other analyses in patient tissues and cybrids were also carried out. We found three new pathologic mutations. Two of them in patients showing phenotypes that have not been commonly associated to mutations in the MT-ATP6 gene. These results remark the importance of sequencing the MT-ATP6 gene in patients with striatal necrosis syndromes, but also within other mitochondrial pathologies. This gene should be sequenced at least in all those patients suspected of suffering an mtDNA disorder disclosing normal results for histochemical and biochemical analyses of respiratory chain.

Description: Immunoglobulin-like domain containing receptor 1 ( ILDR1 ) is a poorly characterized gene that was first identified in lymphoma cells. Recently, ILDR1 has been found to be responsible for autosomal recessive hearing impairment DFNB42. Patients with ILDR1 mutations cause bilateral non-progressive moderate-to-profound sensorineural hearing impairment. However, the etiology and mechanism of ILDR1 -related hearing loss remains to be elucidated. In order to uncover the pathology of DFNB42 deafness, we used the morpholino injection technique to establish an ildr1b -morphant zebrafish model. Ildr1b -morphant zebrafish displayed defective hearing and imbalanced swimming, and developmental delays were seen in the semicircular canals of the inner ear. The gene expression profile and real-time PCR revealed down-regulation of atp1b2b (encoding Na + /K + transporting, beta 2b polypeptide) in ildr1b -morphant zebrafish. We found that injection of atp1b2b mRNA into ildr1b -knockdown zebrafish could rescue the phenotype of developmental delay of the semicircular canals. Moreover, ildr1b -morphant zebrafish had reduced numbers of lateral line neuromasts due to the disruption of lateral line primordium migration. In situ hybridization showed the involvement of attenuated FGF signaling and the chemokine receptor 4b ( cxcr4b ) and chemokine receptor 7b ( cxcr7b ) in posterior lateral line primordium of ildr1b -morphant zebrafish. We concluded that Ildr1b is crucial for the development of the inner ear and the lateral line system. This study provides the first evidence for the mechanism of Ildr1b on hearing in vivo and sheds light on the pathology of DFNB42.

Description: Huntington's disease (HD) is an inherited neurodegenerative disorder caused by abnormal expansion of CAG repeats in the gene encoding huntingtin. Mutant huntingtin undergoes proteolytic processing and its N-terminal fragment containing polyglutamine repeat accumulates as inclusion not only in nucleus but also in cytoplasm and neuronal processes. Here, we demonstrate that removal of ubiquitin ligase Ube3a selectively from HD mice brain resulted in accelerated disease phenotype and shorter lifespan in comparison with HD mice. The deficiency of Ube3a in HD mice brain also caused significant increase in global aggregates load, and these aggregates were less ubiquitinated when compared with age-matched HD mice. These Ube3a -maternal deficient HD mice also showed drastic reduction of DARPP-32, a dopamine-regulated phoshphoprotein in their striatum. These results emphasize the crucial role of Ube3a in the progression of HD and its immense potential as therapeutic target.

Description: Parent-of-origin-specific expression at imprinted genes is regulated by allele-specific DNA methylation at imprinting control regions (ICRs). This mechanism of gene regulation, where one element controls allelic expression of multiple genes, is not fully understood. Furthermore, the mechanism of gene dysregulation through ICR epimutations, such as loss or gain of DNA methylation, remains a mystery. We have used genetic mouse models to dissect ICR-mediated genetic and epigenetic regulation of imprinted gene expression. The H19/insulin-like growth factor 2 (Igf2) ICR has a multifunctional role including insulation, activation and repression. Microdeletions at the human H19/IGF2 ICR (IC1) are proposed to be responsible for IC1 epimutations associated with imprinting disorders such as Beckwith–Wiedemann syndrome (BWS). Here, we have generated and characterized a mouse model that mimics BWS microdeletions to define the role of the deleted sequence in establishing and maintaining epigenetic marks and imprinted expression at the H19/IGF2 locus. These mice carry a 1.3 kb deletion at the H19/Igf2 ICR [2,3] removing two of four CCCTC-binding factor (CTCF) sites and the intervening sequence, ~75% of the ICR. Surprisingly, the 2,3 deletion does not perturb DNA methylation at the ICR; however, it does disrupt imprinted expression. While repressive functions of the ICR are compromised by the deletion regardless of tissue type, insulator function is only disrupted in tissues of mesodermal origin where a significant amount of CTCF is poly(ADP-ribosyl)ated. These findings suggest that insulator activity of the H19/Igf2 ICR varies by cell type and may depend on cell-specific enhancers as well as posttranslational modifications of the insulator protein CTCF.

Description: Cerebral cavernous malformation (CCM) is a disease of vascular malformations known to be caused by mutations in one of three genes: CCM1 , CCM2 or CCM3 . Despite several studies, the mechanism of CCM lesion onset remains unclear. Using a Ccm1 knockout mouse model, we studied the morphogenesis of early lesion formation in the retina in order to provide insight into potential mechanisms. We demonstrate that lesions develop in a stereotypic location and pattern, preceded by endothelial hypersprouting as confirmed in a zebrafish model of disease. The vascular defects seen with loss of Ccm1 suggest a defect in endothelial flow response. Taken together, these results suggest new mechanisms of early CCM disease pathogenesis and provide a framework for further study.

Description: Genomic imprinting is the epigenetic process that results in monoallelic expression of genes depending on parental origin. These genes are known to be critical for placental development and fetal growth in mammals. Aberrant epigenetic profiles at imprinted loci, such as DNA methylation defects, are surprisingly rare in pregnancies with compromised fetal growth, while variations in transcriptional output from the expressed alleles of imprinted genes are more commonly reported in pregnancies complicated with intrauterine growth restriction (IUGR). To determine if PLAGL1 and HYMAI , two imprinted transcripts deregulated in Transient Neonatal Diabetes Mellitus, are involved in non-syndromic IUGR we compared the expression and DNA methylation levels in a large cohort of placental biopsies from IUGR and uneventful pregnancies. This revealed that despite appropriate maternal methylation at the shared PLAGL1 / HYMAI promoter, there was a loss of correlation between PLAGL1 and HYMAI expression in IUGR. This incongruity was due to higher HYMAI expression in IUGR gestations, coupled with PLAGL1 down-regulation in placentas from IUGR girls, but not boys. The PLAGL1 protein is a zinc-finger transcription factor that has been shown to be a master coordinator of a genetic growth network in mice. We observe PLAGL1 binding to the H19 / IGF2 shared enhancers in placentae, with significant correlations between PLAGL1 levels with H19 and IGF2 expression levels. In addition, PLAGL1 binding and expression also correlate with expression levels of metabolic regulator genes SLC2A4 , TCF4 and PPAR1 . Our results strongly suggest that fetal growth can be influenced by altered expression of the PLAGL1 gene network in human placenta.

Description: MicroRNAs (miRNAs) have emerged as a class of small, endogenous, regulatory RNAs that exhibit the ability to epigenetically modulate the translation of mRNAs into proteins. This feature enables them to control cell phenotypes and, consequently, modify cell function in a disease context. The role of inflammatory miRNAs in Alzheimer's disease (AD) and their ability to modulate glia responses are now beginning to be explored. In this study, we propose to disclose the functional role of miR-155, one of the most well studied immune-related miRNAs in AD-associated neuroinflammatory events, employing the 3xTg AD animal model. A strong upregulation of miR-155 levels was observed in the brain of 12-month-old 3xTg AD animals. This event occurred simultaneously with an increase of microglia and astrocyte activation, and before the appearance of extracellular Aβ aggregates, suggesting that less complex Aβ species, such as Aβ oligomers may contribute to early neuroinflammation. In addition, we investigated the contribution of miR-155 and the c-Jun transcription factor to the molecular mechanisms that underlie Aβ-mediated activation of glial cells. Our results suggest early miR-155 and c-Jun upregulation in the 3xTg AD mice, as well as in Aβ-activated microglia and astrocytes, thus contributing to the production of inflammatory mediators such as IL-6 and IFN-β. This effect is associated with a miR-155-dependent decrease of suppressor of cytokine signaling 1. Furthermore, since c-Jun silencing decreases the levels of miR-155 in Aβ-activated microglia and astrocytes, we propose that miR-155 targeting can constitute an interesting and promising approach to control neuroinflammation in AD.

Description: Huntington's disease (HD) is a neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in the huntingtin ( HTT ) gene. Disease pathogenesis derives, at least in part, from the long polyglutamine tract encoded by mutant HTT . Therefore, considerable effort has been dedicated to the development of therapeutic strategies that significantly reduce the expression of the mutant HTT protein. Antisense oligonucleotides (ASOs) targeted to the CAG repeat region of HTT transcripts have been of particular interest due to their potential capacity to discriminate between normal and mutant HTT transcripts. Here, we focus on phosphorodiamidate morpholino oligomers (PMOs), ASOs that are especially stable, highly soluble and non-toxic. We designed three PMOs to selectively target expanded CAG repeat tracts (CTG22, CTG25 and CTG28), and two PMOs to selectively target sequences flanking the HTT CAG repeat (HTTex1a and HTTex1b). In HD patient–derived fibroblasts with expanded alleles containing 44, 77 or 109 CAG repeats, HTTex1a and HTTex1b were effective in suppressing the expression of mutant and non-mutant transcripts. CTGn PMOs also suppressed HTT expression, with the extent of suppression and the specificity for mutant transcripts dependent on the length of the targeted CAG repeat and on the CTG repeat length and concentration of the PMO. PMO CTG25 reduced HTT-induced cytotoxicity in vitro and suppressed mutant HTT expression in vivo in the N171-82Q transgenic mouse model. Finally, CTG28 reduced mutant HTT expression and improved the phenotype of Hdh Q7/Q150 knock-in HD mice. These data demonstrate the potential of PMOs as an approach to suppressing the expression of mutant HTT.

Description: Reduced expression of SMN protein causes spinal muscular atrophy (SMA), a neurodegenerative disorder leading to motor neuron dysfunction and loss. However, the molecular mechanisms by which SMN regulates neuronal dysfunction are not fully understood. Here, we report that reduced SMN protein level alters miRNA expression and distribution in neurons. In particular, miR-183 levels are increased in neurites of SMN-deficient neurons. We demonstrate that miR-183 regulates translation of mTor via direct binding to its 3' UTR. Interestingly, local axonal translation of mTor is reduced in SMN-deficient neurons, and this can be recovered by miR-183 inhibition. Finally, inhibition of miR-183 expression in the spinal cord of an SMA mouse model prolongs survival and improves motor function of Smn -mutant mice. Together, these observations suggest that axonal miRNAs and the mTOR pathway are previously unidentified molecular mechanisms contributing to SMA pathology.

Description: Microphthalmia-associated transcription factor ( MITF ) is a master regulator of pigmented cell survival and differentiation with direct transcriptional links to cell cycle, apoptosis and pigmentation. In mouse, Mitf is expressed early and uniformly in optic vesicle (OV) cells as they evaginate from the developing neural tube, and null Mitf mutations result in microphthalmia and pigmentation defects. However, homozygous mutations in MITF have not been identified in humans; therefore, little is known about its role in human retinogenesis. We used a human embryonic stem cell (hESC) model that recapitulates numerous aspects of retinal development, including OV specification and formation of retinal pigment epithelium (RPE) and neural retina progenitor cells (NRPCs), to investigate the earliest roles of MITF. During hESC differentiation toward a retinal lineage, a subset of MITF isoforms was expressed in a sequence and tissue distribution similar to that observed in mice. In addition, we found that promoters for the MITF-A , -D and -H isoforms were directly targeted by Visual Systems Homeobox 2 (VSX2), a transcription factor involved in patterning the OV toward a NRPC fate. We then manipulated MITF RNA and protein levels at early developmental stages and observed decreased expression of eye field transcription factors, reduced early OV cell proliferation and disrupted RPE maturation. This work provides a foundation for investigating MITF and other highly complex, multi-purposed transcription factors in a dynamic human developmental model system.

Description: The p.N478D missense mutation in human mitochondrial poly(A) polymerase (mtPAP) has previously been implicated in a form of spastic ataxia with optic atrophy. In this study, we have investigated fibroblast cell lines established from family members. The homozygous mutation resulted in the loss of polyadenylation of all mitochondrial transcripts assessed; however, oligoadenylation was retained. Interestingly, this had differential effects on transcript stability that were dependent on the particular species of transcript. These changes were accompanied by a severe loss of oxidative phosphorylation complexes I and IV, and perturbation of de novo mitochondrial protein synthesis. Decreases in transcript polyadenylation and in respiratory chain complexes were effectively rescued by overexpression of wild-type mtPAP. Both mutated and wild-type mtPAP localized to the mitochondrial RNA-processing granules thereby eliminating mislocalization as a cause of defective polyadenylation. In vitro polyadenylation assays revealed severely compromised activity by the mutated protein, which generated only short oligo(A) extensions on RNA substrates, irrespective of RNA secondary structure. The addition of LRPPRC/SLIRP, a mitochondrial RNA-binding complex, enhanced activity of the wild-type mtPAP resulting in increased overall tail length. The LRPPRC/SLIRP effect although present was less marked with mutated mtPAP, independent of RNA secondary structure. We conclude that (i) the polymerase activity of mtPAP can be modulated by the presence of LRPPRC/SLIRP, (ii) N478D mtPAP mutation decreases polymerase activity and (iii) the alteration in poly(A) length is sufficient to cause dysregulation of post-transcriptional expression and the pathogenic lack of respiratory chain complexes.

Description: During postnatal development, neuronal activity controls the remodeling of initially imprecise neuronal connections through the regulation of gene expression. MeCP2 binds to methylated DNA and modulates gene expression during neuronal development and MECP2 mutation causes the autistic disorder Rett syndrome. To investigate a role for MeCP2 in neuronal circuit refinement and to identify activity-dependent MeCP2 transcription regulations, we leveraged the precise organization and accessibility of olfactory sensory axons to manipulation of neuronal activity through odorant exposure in vivo . We demonstrate that olfactory sensory axons failed to develop complete convergence when Mecp2 is deficient in olfactory sensory neurons (OSNs) in an otherwise wild-type animal. Furthermore, we demonstrate that expression of selected adhesion genes was elevated in Mecp2 -deficient glomeruli, while acute odor stimulation in control mice resulted in significantly reduced MeCP2 binding to these gene loci, correlating with increased expression. Thus, MeCP2 is required for both circuitry refinement and activity-dependent transcriptional responses in OSNs.

Description: Simultaneous generation of neural cells and that of the nutrient-supplying vasculature during brain development is called neurovascular coupling. We report on a transgenic mouse with impaired transforming growth factor β (TGFβ)-signalling in forebrain-derived neural cells using a Foxg1-cre knock-in to drive the conditional knock-out of the Tgfbr2 . Although the expression of FOXG1 is assigned to neural progenitors and neurons of the telencephalon, Foxg1 cre/+ ; Tgfbr2 flox/flox (Tgfbr2-cKO) mutants displayed intracerebral haemorrhage. Blood vessels exhibited an atypical, clustered appearance were less in number and displayed reduced branching. Vascular endothelial growth factor (VEGF) A, insulin-like growth factor (IGF) 1, IGF2, TGFβ, inhibitor of DNA binding (ID) 1, thrombospondin (THBS) 2, and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) 1 were altered in either expression levels or tissue distribution. Accordingly, human umbilical vein endothelial cells (HUVEC) displayed branching defects after stimulation with conditioned medium (CM) that was derived from primary neural cultures of the ventral and dorsal telencephalon of Tgfbr2-cKO. Supplementing CM of Tgfbr2-cKO with VEGFA rescued these defects, but application of TGFβ aggravated them. HUVEC showed reduced migration towards CM of mutants compared with controls. Supplementing the CM with growth factors VEGFA, fibroblast growth factor (FGF) 2 and IGF1 partially restored HUVEC migration. In contrast, TGFβ supplementation further impaired migration of HUVEC. We observed differences along the dorso-ventral axis of the telencephalon with regard to the impact of these factors on the phenotype. Together these data establish a TGFBR2-dependent molecular crosstalk between neural and endothelial cells during brain vessel development. These findings will be useful to further elucidate neurovascular interaction in general and to understand pathologies of the blood vessel system such as intracerebral haemorrhages, hereditary haemorrhagic telangiectasia, Alzheimeŕs disease, cerebral amyloid angiopathy or tumour biology.

Description: Pneumoconiosis is the most serious occupational disease in China and its leading cause is occupational silica exposure. Pneumoconiosis takes several years to develop depending on the exposure level of silica. However, individual variation in the susceptibility to pneumoconiosis has been observed among the subjects with similar exposure. We conducted a genome-wide screening with 710 999 single nucleotide polymorphisms (SNPs) in a cohort of 400 coal workers (202 cases and 198 exposed controls) for pneumoconiosis susceptible loci. Seven promising variants were evaluated in an independent cohort of 568 coal workers (323 cases and 245 exposed controls), followed by a second replication on 463 iron ore workers (167 cases and 296 exposed controls). By pooling all of the genome-wide association studies and replication stages together, we found a genome-wide significant ( P 〈 5.0 x 10 –8 ) association for rs73329476 ( P = 1.74 x 10 –8 , OR = 2.17, 95% CI = 1.66–2.85) and two additional replicated associations for rs4320486 ( P 〈 0.05) and rs117626015 ( P 〈 0.05) with combined P -values of 4.29 x 10 –6 and 5.05 x 10 –6 , respectively. In addition, the risk allele T of rs73329476 was significantly associated with lower mRNA expression levels of carboxypeptidase M ( CPM ) in total cellular RNA from whole blood of 156 healthy individuals ( P = 0.0252). The identified pneumoconiosis susceptibility loci may provide new insights into the pathogenesis of pneumoconiosis, and may also have some clinical utility for risk prediction for pneumoconiosis and high-risk population screening for workers with occupational silica exposure.

Description: Hearing function is known to be heritable, but few significant and reproducible associations of genetic variants have been identified to date in the adult population. In this study, genome-wide association results of hearing function from the G-EAR consortium and TwinsUK were used for meta-analysis. Hearing ability in eight population samples of Northern and Southern European ancestry ( n = 4591) and the Silk Road ( n = 348) was measured using pure-tone audiometry and summarized using principal component (PC) analysis. Genome-wide association analyses for PC1–3 were conducted separately in each sample assuming an additive model adjusted for age, sex and relatedness of subjects. Meta-analysis was performed using 2.3 million single-nucleotide polymorphisms (SNPs) tested against each of the three PCs of hearing ability in 4939 individuals. A single SNP lying in intron 6 of the salt-inducible kinase 3 ( SIK3 ) gene was found to be associated with hearing PC2 ( P = 3.7 x 10 –8 ) and further supported by whole-genome sequence in a subset. To determine the relevance of this gene in the ear, expression of the Sik3 protein was studied in mouse cochlea of different ages. Sik3 was expressed in murine hair cells during early development and in cells of the spiral ganglion during early development and adulthood. Our results suggest a developmental role of Sik3 in hearing and may be required for the maintenance of adult auditory function.

Description: Genome instability, epigenetic remodelling and structural chromosomal rearrangements are hallmarks of cancer. However, the coordinated epigenetic effects of constitutional chromosomal rearrangements that disrupt genes associated with congenital neurodevelopmental diseases are poorly understood. To understand the genetic–epigenetic interplay at breakpoints of chromosomal translocations disrupting CG-rich loci, we quantified epigenetic modifications at DLGAP4 ( SAPAP4), a key post-synaptic density 95 (PSD95) associated gene, truncated by the chromosome translocation t(8;20)(p12;q11.23), co-segregating with cerebellar ataxia in a five-generation family. We report significant epigenetic remodelling of the DLGAP4 locus triggered by the t(8;20)(p12;q11.23) translocation and leading to dysregulation of DLGAP4 expression in affected carriers. Disruption of DLGAP4 results in monoallelic hypermethylation of the truncated DLGAP4 promoter CpG island. This induced hypermethylation is maintained in somatic cells of carriers across several generations in a t(8;20) dependent-manner however, is erased in the germ cells of the translocation carriers. Subsequently, chromatin remodelling of the locus-perturbed monoallelic expression of DLGAP4 mRNAs and non-coding RNAs in haploid cells having the translocation. Our results provide new mechanistic insight into the way a balanced chromosomal rearrangement associated with a neurodevelopmental disorder perturbs allele-specific epigenetic mechanisms at breakpoints leading to the deregulation of the truncated locus.

Description: We conducted blinded psychiatric assessments of 26 Amish subjects (52 ± 11 years) from four families with prevalent bipolar spectrum disorder, identified 10 potentially pathogenic alleles by exome sequencing, tested association of these alleles with clinical diagnoses in the larger Amish Study of Major Affective Disorder (ASMAD) cohort, and studied mutant potassium channels in neurons. Fourteen of 26 Amish had bipolar spectrum disorder. The only candidate allele shared among them was rs78247304, a non-synonymous variant of KCNH7 (c.1181G〉A, p.Arg394His). KCNH7 c.1181G〉A and nine other potentially pathogenic variants were subsequently tested within the ASMAD cohort, which consisted of 340 subjects grouped into controls subjects and affected subjects from overlapping clinical categories (bipolar 1 disorder, bipolar spectrum disorder and any major affective disorder). KCNH7 c.1181G〉A had the highest enrichment among individuals with bipolar spectrum disorder ( 2 = 7.3) and the strongest family-based association with bipolar 1 ( P = 0.021), bipolar spectrum ( P = 0.031) and any major affective disorder ( P = 0.016). In vitro, the p.Arg394His substitution allowed normal expression, trafficking, assembly and localization of HERG3/Kv11.3 channels, but altered the steady-state voltage dependence and kinetics of activation in neuronal cells. Although our genome-wide statistical results do not alone prove association, cumulative evidence from multiple independent sources (parallel genome-wide study cohorts, pharmacological studies of HERG-type potassium channels, electrophysiological data) implicates neuronal HERG3/Kv11.3 potassium channels in the pathophysiology of bipolar spectrum disorder. Such a finding, if corroborated by future studies, has implications for mental health services among the Amish, as well as development of drugs that specifically target HERG3/Kv11.3.

Description: Complex III (cytochrome bc 1 ) is a protein complex of the mitochondrial inner membrane that transfers electrons from ubiquinol to cytochrome c . Its assembly requires the coordinated expression of mitochondrial-encoded cytochrome b and nuclear-encoded subunits and assembly factors. Complex III deficiency is a severe multisystem disorder caused by mutations in subunit genes or assembly factors. Sequence-profile-based orthology predicts C11orf83 , hereafter named UQCC3 , to be the ortholog of the fungal complex III assembly factor CBP4 . We describe a homozygous c.59T〉A missense mutation in UQCC3 from a consanguineous patient diagnosed with isolated complex III deficiency, displaying lactic acidosis, hypoglycemia, hypotonia and delayed development without dysmorphic features. Patient fibroblasts have reduced complex III activity and lower levels of the holocomplex and its subunits than controls. They have no detectable UQCC3 protein and have lower levels of cytochrome b protein. Furthermore, in patient cells, cytochrome b is absent from a high-molecular-weight complex III. UQCC3 is reduced in cells depleted for the complex III assembly factors UQCC1 and UQCC2. Conversely, absence of UQCC3 in patient cells does not affect UQCC1 and UQCC2. This suggests that UQCC3 functions in the complex III assembly pathway downstream of UQCC1 and UQCC2 and is consistent with what is known about the function of Cbp4 and of the fungal orthologs of UQCC1 and UQCC2, Cbp3 and Cbp6. We conclude that UQCC3 functions in complex III assembly and that the c.59T〉A mutation has a causal role in complex III deficiency.