ALBERT

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Description: Neurons and other cells require intracellular transport of essential components for viability and function. Previous work has shown that while net amyloid precursor protein (APP) transport is generally anterograde, individual vesicles containing APP move bi-directionally. This discrepancy highlights our poor understanding of the in vivo regulation of APP-vesicle transport. Here, we show that reduction of presenilin (PS) or suppression of gamma-secretase activity substantially increases anterograde and retrograde velocities for APP vesicles. Strikingly, PS deficiency has no effect on an unrelated cargo vesicle class containing synaptotagmin, which is powered by a different kinesin motor. Increased velocities caused by PS or gamma-secretase reduction require functional kinesin-1 and dynein motors. Together, our findings suggest that a normal function of PS is to repress kinesin-1 and dynein motor activity during axonal transport of APP vesicles. Furthermore, our data suggest that axonal transport defects induced by loss of PS-mediated regulatory effects on APP-vesicle motility could be a major cause of neuronal and synaptic defects observed in Alzheimer's Disease (AD) pathogenesis. Thus, perturbations of APP/PS transport could contribute to early neuropathology observed in AD, and highlight a potential novel therapeutic pathway for early intervention, prior to neuronal loss and clinical manifestation of disease.

Description: With age, muscle mass and integrity are progressively lost leaving the elderly frail, weak and unable to independently care for themselves. Defined as sarcopenia, this age-related muscle atrophy appears to be multifactorial but its definite cause is still unknown. Mitochondrial dysfunction has been implicated in this process. Using a novel transgenic mouse model of mitochondrial DNA (mtDNA) double-strand breaks (DSBs) that presents a premature aging-like phenotype, we studied the role of mtDNA damage in muscle wasting. We caused DSBs in mtDNA of adult mice using a ubiquitously expressed mitochondrial-targeted endonuclease, mito- Pst I. We found that a short, transient systemic mtDNA damage led to muscle wasting and a decline in locomotor activity later in life. We found a significant decline in muscle satellite cells, which decreases the muscle's capacity to regenerate and repair during aging. This phenotype was associated with impairment in acetylcholinesterase (AChE) activity and assembly at the neuromuscular junction (NMJ), also associated with muscle aging. Our data suggests that systemic mitochondrial dysfunction plays important roles in age-related muscle wasting by preferentially affecting the myosatellite cell pool.

Description: Spinal muscular atrophy (SMA) is caused by mutations of the survival motor neuron 1 ( SMN1 ) gene, retention of the survival motor neuron 2 ( SMN2 ) gene and insufficient expression of full-length survival motor neuron (SMN) protein. Quinazolines increase SMN2 promoter activity and inhibit the ribonucleic acid scavenger enzyme DcpS. The quinazoline derivative RG3039 has advanced to early phase clinical trials. In preparation for efficacy studies in SMA patients, we investigated the effects of RG3039 in severe SMA mice. Here, we show that RG3039 distributed to central nervous system tissues where it robustly inhibited DcpS enzyme activity, but minimally activated SMN expression or the assembly of small nuclear ribonucleoproteins. Nonetheless, treated SMA mice showed a dose-dependent increase in survival, weight and motor function. This was associated with improved motor neuron somal and neuromuscular junction synaptic innervation and function and increased muscle size. RG3039 also enhanced survival of conditional SMA mice in which SMN had been genetically restored to motor neurons. As this systemically delivered drug may have therapeutic benefits that extend beyond motor neurons, it could act additively with SMN-restoring therapies delivered directly to the central nervous system such as antisense oligonucleotides or gene therapy.

Description: Coronary heart disease (CHD) is the leading cause of death worldwide. Mitochondrial genetic determinant for the development of CHD remains poorly explored. We report there the clinical, genetic, molecular and biochemical characterization of a four-generation Chinese family with maternally inherited CHD. Thirteen of 32 adult members in this family exhibited variable severity and age-at-onset of CHD. Mutational analysis of their mitochondrial genomes identified the tRNA Thr 15927G〉A mutation belonging to the Eastern Asian haplogroup B5. The anticipated destabilization of a highly conserved base-pairing (28C-42G) by the 15927G〉A mutation affects structure and function of tRNA Thr . Northern analysis revealed 80% decrease in the steady-state level of tRNA Thr in the mutant cell lines carrying the 15927G〉A mutation. The 15927G〉A mutation changed the conformation of tRNA Thr , as suggested by slower electrophoretic mobility of mutated tRNA with respect to the wild-type molecule. In addition, ~39% reduction in aminoacylated efficiency of tRNA Thr was observed in mutant cells derived from this Chinese family. An in vivo mitochondrial protein labeling analysis showed ~53% reduction in the rate of mitochondrial translation in mutant cells. The impaired mitochondrial protein synthesis leads to defects in overall respiratory capacity or malate/glutamate-promoted respiration or succinate/glycerol-3-phosphate-promoted respiration, or N,N,N',N '-tetramethyl-pphenylenediamine/ascorbate-promoted respiration in mutant cells. An increasing production of reactive oxygen species was observed in the mutant cells carrying the 15927G〉A mutation. These results provide the direct evidence that the tRNA Thr 15927G〉A mutation is associated with CHD. Our findings may provide new insights into pathophysiology and intervention targets of this disorder.

Description: Spinal muscular atrophy (SMA) is a devastating neuromuscular disorder that stems from low levels of survival of motor neuron (SMN) protein. The processes that cause motor neurons and muscle cells to become dysfunctional are incompletely understood. We are interested in neuromuscular homeostasis and the stresses put upon that system by loss of SMN. We recently reported that α-COP, a member of the coatomer complex of coat protein I (COPI) vesicles, is an SMN-binding partner, implicating this protein complex in normal SMN function. To investigate the functional significance of the interaction between α-COP and SMN, we constructed an inducible NSC-34 cell culture system to model the consequences of SMN depletion and find that depletion of SMN protein results in shortened neurites. Heterologous expression of human SMN, and interestingly over-expression of α-COP, restores normal neurite length and morphology. Mutagenesis of the canonical COPI dilysine motifs in exon 2b results in failure to bind to α-COP and abrogates the ability of human SMN to restore neurite outgrowth in SMN-depleted motor neuron-like NSC-34 cells. We conclude that the interaction between SMN and α-COP serves an important function in the growth and maintenance of motor neuron processes and may play a significant role in the pathogenesis of SMA.

Description: Human cortical malformations, including lissencephaly, polymicrogyria and other diseases of neurodevelopment, have been associated with mutations in microtubule subunits and microtubule-associated proteins. Here we report our cloning of the brain dimple ( brdp ) mouse mutation, which we recovered from an ENU screen for recessive perinatal phenotypes affecting neurodevelopment. We identify the causal mutation in the tubulin, beta-2b ( Tubb2b) gene as a missense mutation at a highly conserved residue (N247S). Brdp/brdp homozygous mutants have significant thinning of the cortical epithelium, which is markedly more severe in the caudo-lateral portion of the telencephalon, and do not survive past birth. The cortical defects are largely due to a major increase in apoptosis and we note abnormal proliferation of the basal progenitors. Adult brdp/+ mice are viable and fertile but exhibit behavioral phenotypes. This allele of Tubb2b represents the most severely affected mouse tubulin phenotype reported to date and this is the first report of a tubulin mutation affecting neuronal proliferation and survival.

Description: CCDC28B encodes a coiled coil domain-containing protein involved in ciliogenesis that was originally identified as a second site modifier of the ciliopathy Bardet–Biedl syndrome. We have previously shown that the depletion of CCDC28B leads to shortened cilia; however, the mechanism underlying how this protein controls ciliary length is unknown. Here, we show that CCDC28B interacts with SIN1, a component of the mTOR complex 2 (mTORC2), and that this interaction is important both in the context of mTOR signaling and in a hitherto unknown, mTORC-independent role of SIN1 in cilia biology. We show that CCDC28B is a positive regulator of mTORC2, participating in its assembly/stability and modulating its activity, while not affecting mTORC1 function. Further, we show that Ccdc28b regulates cilia length in vivo , at least in part, through its interaction with Sin1. Importantly, depletion of Rictor, another core component of mTORC2, does not result in shortened cilia. Taken together, our findings implicate CCDC28B in the regulation of mTORC2, and uncover a novel function of SIN1 regulating cilia length that is likely independent of mTOR signaling.

Description: Spinal muscular atrophy (SMA) is caused by insufficient levels of the survival motor neuron (SMN) protein due to the functional loss of the SMN1 gene and the inability of its paralog, SMN2 , to fully compensate due to reduced exon 7 splicing efficiency. Since SMA patients have at least one copy of SMN2 , drug discovery campaigns have sought to identify SMN2 inducers. C5-substituted quinazolines increase SMN2 promoter activity in cell-based assays and a derivative, RG3039, has progressed to clinical testing. It is orally bioavailable, brain-penetrant and has been shown to be an inhibitor of the mRNA decapping enzyme, DcpS. Our pharmacological characterization of RG3039, reported here, demonstrates that RG3039 can extend survival and improve function in two SMA mouse models of varying disease severity (Taiwanese 5058 Hemi and 2B/– SMA mice), and positively impacts neuromuscular pathologies. In 2B/– SMA mice, RG3039 provided a 〉600% survival benefit (median 18 days to 〉112 days) when dosing began at P4, highlighting the importance of early intervention. We determined the minimum effective dose and the associated pharmacokinetic (PK) and exposure relationship of RG3039 and DcpS inhibition ex vivo . These data support the long PK half-life with extended pharmacodynamic outcome of RG3039 in 2B/– SMA mice. In motor neurons, RG3039 significantly increased both the average number of cells with gems and average number of gems per cell, which is used as an indirect measure of SMN levels. These studies contribute to dose selection and exposure estimates for the first studies with RG3039 in human subjects.

Description: Amyotrophic lateral sclerosis (ALS) is a devastating neurological disorder characterized by selective degeneration of upper and lower motoneurons. The primary triggers for motoneuron degeneration are still unknown, but inflammation is considered an important contributing factor. P2X7 receptor is a key player in microglia response to toxic insults and was previously shown to increase pro-inflammatory actions of SOD1-G93A ALS microglia. We therefore hypothesized that lack of P2X7 receptor could modify disease features in the SOD1-G93A mice. Hetero- and homozygous P2X7 receptor knock-out SOD1-G93A mice were thus generated and analysed for body weight, disease onset and progression (by behavioural scores, grip and rotarod tests) and survival. Although the lifespan of P2X7 +/– and P2X7 –/– /SOD1-G93A female mice was extended by 6–7% with respect to SOD1-G93A mice, to our surprise the clinical onset was significantly anticipated and the disease progression worsened in both male and female P2X7 –/– /SOD1-G93A mice. Consistently, we found increased astrogliosis, microgliosis, motoneuron loss, induction of the pro-inflammatory markers NOX2 and iNOS and activation of the MAPKs pathway in the lumbar spinal cord of end-stage P2X7 –/– /SOD1-G93A mice. These results show that the constitutive deletion of P2X7 receptor aggravates the ALS pathogenesis, suggesting that the receptor might have beneficial effects in at least definite stages of the disease. This study unravels a complex dual role of P2X7 receptor in ALS and strengthens the importance of a successful time window of therapeutic intervention in contrasting the pathology.

Description: There are certain de novo germline mutations associated with genetic disorders whose mutation rates per generation are orders of magnitude higher than the genome average. Moreover, these mutations occur exclusively in the male germ line and older men have a higher probability of having an affected child than younger ones, known as the paternal age effect (PAE). The classic example of a genetic disorder exhibiting a PAE is achondroplasia, caused predominantly by a single-nucleotide substitution (c.1138G〉A) in FGFR3 . To elucidate what mechanisms might be driving the high frequency of this mutation in the male germline, we examined the spatial distribution of the c.1138G〉A substitution in a testis from an 80-year-old unaffected man. Using a technology based on bead-emulsion amplification, we were able to measure mutation frequencies in 192 individual pieces of the dissected testis with a false-positive rate lower than 2.7 x 10 –6 . We observed that most mutations are clustered in a few pieces with 95% of all mutations occurring in 27% of the total testis. Using computational simulations, we rejected the model proposing an elevated mutation rate per cell division at this nucleotide site. Instead, we determined that the observed mutation distribution fits a germline selection model, where mutant spermatogonial stem cells have a proliferative advantage over unmutated cells. Combined with data on several other PAE mutations, our results support the idea that the PAE, associated with a number of Mendelian disorders, may be explained primarily by a selective mechanism.

Description: microRNAs (miRNAs) are dysregulated in a variety of disease states, suggesting that this newly discovered class of gene expression repressors may be viable therapeutic targets. A microarray of miRNA changes in ALS-model superoxide dismutase 1 (SOD1) G93A rodents identified 12 miRNAs as significantly changed. Six miRNAs tested in human ALS tissues were confirmed increased. Specifically, miR-155 was increased 5-fold in mice and 2-fold in human spinal cords. To test miRNA inhibition in the central nervous system (CNS) as a potential novel therapeutic, we developed oligonucleotide-based miRNA inhibitors (anti-miRs) that could inhibit miRNAs throughout the CNS and in the periphery. Anti-miR-155 caused global derepression of targets in peritoneal macrophages and, following intraventricular delivery, demonstrated widespread functional distribution in the brain and spinal cord. After treating SOD1 G93A mice with anti-miR-155, we significantly extended survival by 10 days and disease duration by 15 days (38%) while a scrambled control anti-miR did not significantly improve survival or disease duration. Therefore, antisense oligonucleotides may be used to successfully inhibit miRNAs throughout the brain and spinal cord, and miR-155 is a promising new therapeutic target for human ALS.

Description: Multiple sclerosis (MS) is the most common autoimmune disease of the central nervous system (CNS). It is characterized by the infiltration of autoreactive immune cells into the CNS, which target the myelin sheath, leading to the loss of neuronal function. Although it is accepted that MS is a multifactorial disorder with both genetic and environmental factors influencing its development and course, the molecular pathogenesis of MS has not yet been fully elucidated. Here, we studied the longitudinal gene expression profiles of whole-blood RNA from a cohort of 195 MS patients and 66 healthy controls. We analyzed these transcriptomes at both the individual transcript and the biological pathway level. We found 62 transcripts to be significantly up-regulated in MS patients; the expression of 11 of these genes was counter-regulated by interferon treatment, suggesting partial restoration of a ‘healthy’ gene expression profile. Global pathway analyses linked the proteasome and Wnt signaling to MS disease processes. Since genotypes from a subset of individuals were available, we were able to identify expression quantitative trait loci (eQTL), a number of which involved two genes of the MS gene signature. However, all these eQTL were also present in healthy controls. This study highlights the challenge posed by analyzing transcripts from whole blood and how these can be mitigated by using large, well-characterized cohorts of patients with longitudinal follow-up and multi-modality measurements.

Description: Cornelia de Lange syndrome (CdLS) is a developmental disorder caused by mutations in NIPBL, a protein which has functionally been associated with the cohesin complex. Mutations in core cohesin complex components have also been reported in individuals with CdLS-like phenotypes. In addition to its role in sister chromatid cohesion, cohesin is thought to play a role in regulating gene expression during development. The mechanism of this gene regulation remains unclear, but NIPBL and cohesin have been reported to affect long-range chromosomal interactions, both independently and through interactions with CTCF. We used fluorescence in situ hybridization to investigate whether the disruption of NIPBL affects chromosome architecture. We show that cells from CdLS patients exhibit visible chromatin decompaction, that is most pronounced across gene-rich regions of the genome. Cells carrying mutations predicted to have a more severe effect on NIPBL function show more extensive chromatin decompaction than those carrying milder mutations. This cellular phenotype was reproduced in normal cells depleted for NIPBL with siRNA, but was not seen following the knockdown of either the cohesin component SMC3, or CTCF. We conclude that NIPBL has a function in modulating chromatin architecture, particularly for gene-rich areas of the chromosome, that is not dependent on SMC3/cohesin or CTCF, raising the possibility that the aetiology of disorders associated with the mutation of core cohesin components is distinct from that associated with the disruption of NIPBL itself in classical CdLS.

Description: Skin barrier function is primarily assigned to the outer epidermal layer, the stratum corneum (SC), mainly composed of corneocytes and lipid-enriched extracellular matrix. Epidermal ceramides (Cers) are essential barrier lipids, containing ultra-long-chain (ULC) fatty acids (FAs) with a unique -hydroxy group, which is necessary for binding to corneocyte proteins. In the SC, Cers are believed to derive from glucosylated intermediates, namely glucosylceramides (GlcCers), as surmised from human Gaucher's disease and related mouse models. Tamoxifen (TAM)-induced deletion of the endogenous GlcCer-synthesizing enzyme UDP-glucose:ceramide glucosyltransferase (UGCG) in keratin K14-positive cells resulted in epidermal GlcCer depletion. Although free extractable Cers were elevated in total epidermis and as well in SC, protein-bound Cers decreased significantly in Ugcg f/fK14CreERT2 mice, indicating glucosylation to be required for regular Cer processing as well as arrangement and extrusion of lipid lamellae. The almost complete loss of protein-bound Cers led to a disruption of the water permeability barrier (WPB). UGCG-deficient mice developed an ichthyosis-like skin phenotype marked by impaired keratinocyte differentiation associated with delayed wound healing. Gene expression profiling of Ugcg -mutant skin revealed a subset of differentially expressed genes involved in lipid signaling and epidermal differentiation/proliferation, correlating to human skin diseases such as psoriasis and atopic dermatitis. Peroxisome proliferator-activated receptor beta/delta (PPARβ/), a Cer-sensitive transcription factor was identified as potential mediator of the altered gene sets.

Description: A long-standing pathomechanistic model proposes that the polyglutamine (polyQ)-length-dependent toxicity threshold observed in all polyQ diseases is triggered by a conformational change within the monomer that occurs only above a certain polyQ length. If true, this yet undefined and elusive mutant-specific toxic conformation would constitute a direct therapeutic target. Three anti-polyQ antibodies—MW1, 1C2 and 3B5H10—have been extensively used to probe the conformation of polyQ. The crystal structure of the MW1 epitope reveals a linear, non-pathogenic polyQ. In contrast, although the detailed structure of its epitope is unknown, the 3B5H10 antibody is widely advertised and used as a conformational antibody that recognizes the toxic conformation of expanded polyQ. We solved the crystal structure of the 1C2 antigen-binding domain (1C2-Fab) and performed a direct comparison between the 1C2, MW1 and 3B5H10 structures. The MW1 and 1C2 antibodies have similar sequences and structures, consistent with their binding to short polyQ and their polyQ length-discrimination properties. Unexpectedly, the 3B5H10 antibody also shares striking features with MW1 and 1C2, which prompted us to revisit its binding properties. We show that the 3B5H10 epitope is actually a short, non-pathogenic polyQ. All three antibodies MW1, 1C2 and 3B5H10 interact similarly with polyQ of various lengths, and bind small polyQ epitopes in similar linear and extended conformations. Together with studies published during the recent years, our work argues against the hypothesis that a mutant-specific conformation in monomeric polyQ molecules is the toxic entity responsible for polyQ diseases.

Description: Charcot–Marie–Tooth disease (CMT) comprises a clinically and genetically heterogeneous group of peripheral neuropathies characterized by progressive distal muscle weakness and atrophy, foot deformities and distal sensory loss. Following the analysis of two consanguineous families affected by a medium to late-onset recessive form of intermediate CMT, we identified overlapping regions of homozygosity on chromosome 1p36 with a combined maximum LOD score of 5.4. Molecular investigation of the genes from this region allowed identification of two homozygous mutations in PLEKHG5 that produce premature stop codons and are predicted to result in functional null alleles. Analysis of Plekhg5 in the mouse revealed that this gene is expressed in neurons and glial cells of the peripheral nervous system, and that knockout mice display reduced nerve conduction velocities that are comparable with those of affected individuals from both families. Interestingly, a homozygous PLEKHG5 missense mutation was previously reported in a recessive form of severe childhood onset lower motor neuron disease (LMND) leading to loss of the ability to walk and need for respiratory assistance. Together, these observations indicate that different mutations in PLEKHG5 lead to clinically diverse outcomes (intermediate CMT or LMND) affecting the function of neurons and glial cells.

Description: Disappearance of TAR-DNA-binding protein 43 kDa (TDP-43) from the nucleus contributes to the pathogenesis of amyotrophic lateral sclerosis (ALS), but the nuclear function of TDP-43 is not yet fully understood. TDP-43 associates with nuclear bodies including Gemini of coiled bodies (GEMs). GEMs contribute to the biogenesis of uridine-rich small nuclear RNA (U snRNA), a component of splicing machinery. The number of GEMs and a subset of U snRNAs decrease in spinal muscular atrophy, a lower motor neuron disease, suggesting that alteration of U snRNAs may also underlie the molecular pathogenesis of ALS. Here, we investigated the number of GEMs and U11/12-type small nuclear ribonucleoproteins (snRNP) by immunohistochemistry and the level of U snRNAs using real-time quantitative RT-PCR in ALS tissues. GEMs decreased in both TDP-43-depleted HeLa cells and spinal motor neurons in ALS patients. Levels of several U snRNAs decreased in TDP-43-depleted SH-SY5Y and U87-MG cells. The level of U12 snRNA was decreased in tissues affected by ALS (spinal cord, motor cortex and thalamus) but not in tissues unaffected by ALS (cerebellum, kidney and muscle). Immunohistochemical analysis revealed the decrease in U11/12-type snRNP in spinal motor neurons of ALS patients. These findings suggest that loss of TDP-43 function decreases the number of GEMs, which is followed by a disturbance of pre-mRNA splicing by the U11/U12 spliceosome in tissues affected by ALS.

Description: Uromodulin-associated kidney disease (UAKD) is a dominant heritable renal disease in humans which is caused by mutations in the uromodulin ( UMOD) gene and characterized by heterogeneous clinical appearance. To get insights into possible causes of this heterogeneity of UAKD, we describe the new mutant mouse line Umod C93F , leading to disruption of a putative disulfide bond which is also absent in a known human UMOD mutation, and compare the phenotype of this new mouse line with the recently published mouse line Umod A227T . In both mutant mouse lines, which were both bred on the C3H background, the Umod mutations cause a gain-of-toxic function due to a maturation defect of the mutant uromodulin leading to a dysfunction of thick ascending limb of Henle's loop (TALH) cells of the kidney. Umod mutant mice exhibit increased plasma urea and Cystatin levels, impaired urinary concentration ability, reduced fractional excretion of uric acid and nephropathological alterations including uromodulin retention in TALH cells, interstitial fibrosis and inflammatory cell infiltrations, tubular atrophy and occasional glomerulo- und tubulocystic changes, a phenotype highly similar to UAKD in humans. The maturation defect of mutant uromodulin leads to the accumulation of immature uromodulin in the endoplasmic reticulum (ER) and to ER hyperplasia. Further, this study was able to demonstrate for the first time in vivo that the severity of the uromodulin maturation defect as well as onset and speed of progression of renal dysfunction and morphological alterations are strongly dependent on the particular Umod mutation itself and the zygosity status.

Description: Hepatitis B virus (HBV) infection is the predominant risk factor for chronic hepatitis B (CHB), liver cirrhosis (LC) and hepatocellular carcinoma (HCC). Recently, several genome-wide association studies (GWASs) of CHB identified human leukocyte antigen (HLA) loci, including HLA-DP and HLA-DQ in Asian populations, as being associated with the risk of CHB. To confirm and identify the host genetic factors related to CHB infection, we performed another GWAS using a higher-density chip in Korean CHB carriers. We analyzed 1400 samples from Korean population (400 CHB cases and 1000 population controls) using a higher-density GWAS chip [1 140 419 single nucleotide polymorphisms (SNPs)]. In subsequent replication analysis, we further analyzed in an independent study of a Korean CHB cohort consisting of 2909 Korean samples (971 cases and 1938 controls). Logistic regression methods were used for statistical analysis adjusting for age and sex as covariates. This study identified two new risk-associated loci for CHB on the HLA region of chromosome 6, e.g. rs652888 on euchromatic histone-lysine-methyltransferase 2 (EHMT2, P = 7.07 x 10 –13 ) and rs1419881 on transcription factor 19 (TCF19, P = 1.26 x 10 –18 ). Conditional analysis with nearby HLA CHB loci that were previously known, confirmed the independent genetic effects of these two loci on CHB. Conclusion : The GWAS and the subsequent validation study identified new variants associated with the risk of CHB. These findings may advance the understanding of genetic susceptibility to CHB.

Description: Facio-scapulo-humeral dystrophy (FSHD) results from deletions in the subtelomeric macrosatellite D4Z4 array on the 4q35 region. Upregulation of the DUX4 retrogene from the last D4Z4 repeated unit is thought to underlie FSHD pathophysiology. However, no one knows what triggers muscle defect and when alteration arises. To gain further insights into the molecular mechanisms of the disease, we evaluated at the molecular level, the perturbation linked to the FSHD genotype with no a priori on disease onset, severity or penetrance and prior to any infiltration by fibrotic or adipose tissue in biopsies from fetuses carrying a short pathogenic D4Z4 array ( n = 6) compared with fetuses with a non-pathogenic D4Z4 array ( n = 21). By measuring expression of several muscle-specific markers and 4q35 genes including the DUX4 retrogene by an RT-PCR and western blotting, we observed a global dysregulation of genes involved in myogenesis including MYOD1 in samples with 〈11 D4Z4 . The DUX4-fl pathogenic transcript was detected in FSHD biopsies but also in controls. Importantly, in FSHD fetuses, we mainly detected the non-spliced DUX4-fl isoform. In addition, several other genes clustered at the 4q35 locus are upregulated in FSHD fetuses. Our study is the first to examine fetuses carrying an FSHD-linked genotype and reveals an extensive dysregulation of several muscle-specific and 4q35 genes at early development stage at a distance from any muscle defect. Overall, our work suggests that even if FSHD is an adult-onset muscular dystrophy, the disease might also involve early molecular defects arising during myogenesis or early differentiation.

Description: Two siblings from consanguineous parents died perinatally with a condition characterized by generalized hypotonia, respiratory insufficiency, arthrogryposis, microcephaly, congenital brain malformations and hyperglycinemia. Catalytic activities of the mitochondrial respiratory complexes I and II were deficient in skeletal muscle, a finding suggestive of an inborn error in mitochondrial biogenesis. Homozygosity mapping identified IBA57 located in the largest homozygous region on chromosome 1 as a culprit candidate gene. IBA57 is known to be involved in the biosynthesis of mitochondrial [4Fe-4S] proteins. Sequence analysis of IBA57 revealed the homozygous mutation c.941A 〉 C, p.Gln314Pro. Severely decreased amounts of IBA57 protein were observed in skeletal muscle and cultured skin fibroblasts from the affected subjects. HeLa cells depleted of IBA57 showed biochemical defects resembling the ones found in patient-derived cells, including a decrease in various mitochondrial [4Fe-4S] proteins and in proteins covalently linked to lipoic acid (LA), a cofactor produced by the [4Fe-4S] protein LA synthase. The defects could be complemented by wild-type IBA57 and partially by mutant IBA57 . As a result of the mutation, IBA57 protein was excessively degraded, an effect ameliorated by protease inhibitors. Hence, we propose that the mutation leads to partial functional impairment of IBA57, yet the major pathogenic impact is due to its proteolytic degradation below physiologically critical levels. In conclusion, the ensuing lethal complex biochemical phenotype of a novel metabolic syndrome results from multiple Fe/S protein defects caused by a deficiency in the Fe/S cluster assembly protein IBA57.

Description: Duchenne muscular dystrophy (DMD) is characterized by severe degeneration and necrosis of both skeletal and cardiac muscle. While many experimental therapies have shown great promise in treating skeletal muscle disease, an effective therapy for Duchenne cardiomyopathy remains a challenge in large animal models and human patients. The current views on cardiac consequences of skeletal muscle-centered therapy are controversial. Studies performed in young adult mdx mice (a mild DMD mouse model) have yielded opposing results. Since mdx mice do not develop dystrophic cardiomyopathy until ≥21 months of age, we reasoned that old mdx mice may represent a better model to assess the impact of skeletal muscle rescue on dystrophic heart disease. Here, we aged skeletal muscle-specific micro-dystrophin transgenic mdx mice to 23 months and examined the cardiac phenotype. As expected, transgenic mdx mice had minimal skeletal muscle disease and they also outperformed original mdx mice on treadmill running. On cardiac examination, the dystrophin-null heart of transgenic mdx mice displayed severe cardiomyopathy matching that of non-transgenic mdx mice. Specifically, both the strains showed similar heart fibrosis and cardiac function deterioration in systole and diastole. Cardiac output and ejection fraction were also equally compromised. Our results suggest that skeletal muscle rescue neither aggravates nor alleviates cardiomyopathy in aged mdx mice. These findings underscore the importance of treating both skeletal and cardiac muscles in DMD therapy.

Description: Mutations in Parkin or PINK1 are the most common cause of recessively inherited parkinsonism. Parkin and PINK1 function in a conserved mitochondrial quality control pathway, in which PINK1, a putative mitochondrial kinase, directs Parkin, a cytosolic E3 ubiquitin ligase, selectively to dysfunctional mitochondria to promote their isolation, immobilization and degradation by macroautophagy (hereafter, mitophagy). As Parkin recruitment to mitochondria is robustly induced by PINK1 expression on the outer mitochondrial membrane, Parkin recruitment to mitochondria was used as an assay for PINK1 function. Unexpectedly, mutation of serine residues within the activation segment of PINK1 uncovered a temperature-sensitive variant of PINK1 (tsPINK1). tsPINK1 allowed for the first time the disassociation of PINK1 activity from its expression and localization. Additionally, extensive mutagenesis identified three disease-associated variants in the activation segment and one in an α-helix N-terminal to kinase domain (Q126P) that are similarly thermally labile, suggesting that their activity could be restored post-translationally (e.g. by reducing the temperature or by a chemical or pharmacologic chaperone). Together, these findings suggest that tsPINK1 may represent a valuable tool for the analysis of the PINK1/Parkin pathway in human cells; additionally, as the serine residue promoting thermal lability is conserved among Mus musculus , Danio rerio , Drosophila melanogaster and Caenorhabditis elegans , it may serve as the basis for developing other temperature-sensitive models for the study of recessive parkinsonism and mitophagy. Finally, these results suggest that PINK1 kinase function could be restored for a subset of patients with PINK1 mutations, and perhaps alter the course of their disease.