Showing papers in "Human Molecular Genetics in 2017"

Genetics of glaucoma.

Abstract: Genetic and genomic studies, including genome-wide association studies (GWAS) have accelerated the discovery of genes contributing to glaucoma, the leading cause of irreversible blindness world-wide. Glaucoma can occur at all ages, with Mendelian inheritance typical for the rare early onset disease (before age 40) and complex inheritance evident in common adult-onset forms of disease. Recent studies have suggested possible therapeutic targets for some patients with early-onset glaucoma based on the molecular and cellular events caused by MYOC, OPTN and TBK1 mutations. Diagnostic genetic tests using early-onset glaucoma genes are also proving useful for pre-symptomatic disease detection and genetic counseling. Recent GWAS completed for three types of common adult-onset glaucoma have identified novel loci for POAG (primary-open-angle glaucoma) (ABCA1, AFAP1, GMDS, PMM2, TGFBR3, FNDC3B, ARHGEF12, GAS7, FOXC1, ATXN2, TXNRD2); PACG (primary angle-closure glaucoma (EPDR1, CHAT, GLIS3, FERMT2, DPM2-FAM102); and exfoliation syndrome (XFS) glaucoma (CACNA1A). In total sixteen genomic regions have been associated with POAG (including the normal tension glaucoma (NTG) subgroup), 8 with PACG and 2 with XFS. These studies are defining important biological pathways and processes that contribute to disease pathogenesis.

Fat Mass and Obesity-associated (FTO) Protein Regulates Adult Neurogenesis.

Abstract: Fat mass and obesity-associated gene (FTO) is a member of the Fe (II)- and oxoglutarate-dependent AlkB dioxygenase family and is linked to both obesity and intellectual disability. The role of FTO in neurodevelopment and neurogenesis, however, remains largely unknown. Here we show that FTO is expressed in adult neural stem cells and neurons and displays dynamic expression during postnatal neurodevelopment. The loss of FTO leads to decreased brain size and body weight. We find that FTO deficiency could reduce the proliferation and neuronal differentiation of adult neural stem cells in vivo, which leads to impaired learning and memory. Given the role of FTO as a demethylase of N6-methyladenosine (m6A), we went on to perform genome-wide m6A profiling and observed dynamic m6A modification during postnatal neurodevelopment. The loss of FTO led to the altered expression of several key components of the brain derived neurotrophic factor pathway that were marked by m6A. These results together suggest FTO plays important roles in neurogenesis, as well as in learning and memory.

Maternal BMI at the start of pregnancy and offspring epigenome-wide DNA methylation: findings from the pregnancy and childhood epigenetics (PACE) consortium

Abstract: Pre-pregnancy maternal obesity is associated with adverse offspring outcomes at birth and later in life. Individual studies have shown that epigenetic modifications such as DNA methylation could contribute. Within the Pregnancy and Childhood Epigenetics (PACE) Consortium, we meta-analysed the association between pre-pregnancy maternal BMI and methylation at over 450,000 sites in newborn blood DNA, across 19 cohorts (9,340 mother-newborn pairs). We attempted to infer causality by comparing the effects of maternal versus paternal BMI and incorporating genetic variation. In four additional cohorts (1,817 mother-child pairs), we meta-analysed the association between maternal BMI at the start of pregnancy and blood methylation in adolescents. In newborns, maternal BMI was associated with small (<0.2% per BMI unit (1 kg/m2), P < 1.06 × 10-7) methylation variation at 9,044 sites throughout the genome. Adjustment for estimated cell proportions greatly attenuated the number of significant CpGs to 104, including 86 sites common to the unadjusted model. At 72/86 sites, the direction of the association was the same in newborns and adolescents, suggesting persistence of signals. However, we found evidence for acausal intrauterine effect of maternal BMI on newborn methylation at just 8/86 sites. In conclusion, this well-powered analysis identified robust associations between maternal adiposity and variations in newborn blood DNA methylation, but these small effects may be better explained by genetic or lifestyle factors than a causal intrauterine mechanism. This highlights the need for large-scale collaborative approaches and the application of causal inference techniques in epigenetic epidemiology.

Association analyses of East Asian individuals and trans-ancestry analyses with European individuals reveal new loci associated with cholesterol and triglyceride levels.

Abstract: Large-scale meta-analyses of genome-wide association studies (GWAS) have identified >175 loci associated with fasting cholesterol levels, including total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and triglycerides (TG). With differences in linkage disequilibrium (LD) structure and allele frequencies between ancestry groups, studies in additional large samples may detect new associations. We conducted staged GWAS meta-analyses in up to 69,414 East Asian individuals from 24 studies with participants from Japan, the Philippines, Korea, China, Singapore, and Taiwan. These meta-analyses identified (P < 5 × 10-8) three novel loci associated with HDL-C near CD163-APOBEC1 (P = 7.4 × 10-9), NCOA2 (P = 1.6 × 10-8), and NID2-PTGDR (P = 4.2 × 10-8), and one novel locus associated with TG near WDR11-FGFR2 (P = 2.7 × 10-10). Conditional analyses identified a second signal near CD163-APOBEC1. We then combined results from the East Asian meta-analysis with association results from up to 187,365 European individuals from the Global Lipids Genetics Consortium in a trans-ancestry meta-analysis. This analysis identified (log10Bayes Factor ≥6.1) eight additional novel lipid loci. Among the twelve total loci identified, the index variants at eight loci have demonstrated at least nominal significance with other metabolic traits in prior studies, and two loci exhibited coincident eQTLs (P < 1 × 10-5) in subcutaneous adipose tissue for BPTF and PDGFC. Taken together, these analyses identified multiple novel lipid loci, providing new potential therapeutic targets.

Cell-type deconvolution from DNA methylation: a review of recent applications.

Abstract: Recent advances in cell-type deconvolution approaches are adding to our understanding of the biology underlying disease development and progression. DNA methylation (DNAm) can be used as a biomarker of cell types, and through deconvolution approaches, to infer underlying cell type proportions. Cell-type deconvolution algorithms have two main categories: reference-based and reference-free. Reference-based algorithms are supervised methods that determine the underlying composition of cell types within a sample by leveraging differentially methylated regions (DMRs) specific to cell type, identified from DNAm measures of purified cell populations. Reference-free algorithms are unsupervised methods for use when cell-type specific DMRs are not available, allowing scientists to estimate putative cellular proportions or control for potential confounding from cell type. Reference-based deconvolution is typically applied to blood samples and has potentiated our understanding of the relation between immune profiles and disease by allowing estimation of immune cell proportions from archival DNA. Bioinformatic analyses using DNAm to infer immune cell proportions, part of a new field known as Immunomethylomics, provides a new direction for consideration in epigenome wide association studies (EWAS).

APOE ε4/ε4 diminishes neurotrophic function of human iPSC-derived astrocytes.

Abstract: The e4 allele of the APOE gene encoding apolipoprotein E (apoE) is a strong genetic risk factor for aging-related cognitive decline as well as late-onset Alzheimer's disease (AD) compared to the common e3 allele. In the central nervous system, apoE is produced primarily by astrocytes and functions in transporting lipids including cholesterol to support neuronal homeostasis and synaptic integrity. Although mouse models and corresponding primary cells have provided valuable tools for studying apoE isoform-dependent functions, recent studies have shown that human astrocytes have a distinct gene expression profile compare with rodent astrocytes. Human induced pluripotent stem cells (iPSCs) derived from individuals carrying specific gene variants or mutations provide an alternative cellular model more relevant to humans upon differentiation into specific cell types. Thus, we reprogramed human skin fibroblasts from cognitively normal individuals carrying APOE e3/e3 or e4/e4 genotype to iPSC clones and further differentiated them into neural progenitor cells and then astrocytes. We found that human iPSC-derived astrocytes secreted abundant apoE with apoE4 lipoprotein particles less lipidated compared to apoE3 particles. More importantly, human iPSC-derived astrocytes were capable of promoting neuronal survival and synaptogenesis when co-cultured with iPSC-derived neurons with APOE e4/e4 astrocytes less effective in supporting these neurotrophic functions than those with APOE e3/e3 genotype. Taken together, our findings demonstrate APOE genotype-dependent effects using human iPSC-derived astrocytes and provide novel evidence that the human iPSC-based model system is a strong tool to explore how apoE isoforms contribute to neurodegenerative diseases.

The genetics of circadian rhythms, sleep and health

Abstract: Circadian rhythms are 24-h rhythms in physiology and behaviour generated by molecular clocks, which serve to coordinate internal time with the external world. The circadian system is a master regulator of nearly all physiology and its disruption has major consequences on health. Sleep and circadian rhythm disruption (SCRD) is a ubiquitous feature in today's 24/7 society, and studies on shift-workers have shown that SCRD can lead not only to cognitive impairment, but also metabolic syndrome and psychiatric illness including depression (1,2). Mouse models of clock mutants recapitulate these deficits, implicating mechanistic and causal links between SCRD and disease pathophysiology (3-5). Importantly, treating clock disruption reverses and attenuates these adverse health states in animal models (6,7), thus establishing the circadian system as a novel therapeutic target. Significantly, circadian and clock-controlled gene mutations have recently been identified by Genome-Wide Association Studies (GWAS) in the aetiology of sleep, mental health and metabolic disorders. This review will focus upon the genetics of circadian rhythms in sleep and health.

Absence of RNase H2 triggers generation of immunogenic micronuclei removed by autophagy

Abstract: Hypomorphic mutations in the DNA repair enzyme RNase H2 cause the neuroinflammatory autoimmune disorder Aicardi-Goutieres syndrome (AGS). Endogenous nucleic acids are believed to accumulate in patient cells and instigate pathogenic type I interferon expression. However, the underlying nucleic acid species amassing in the absence of RNase H2 has not been established yet. Here, we report that murine RNase H2 knockout cells accumulated cytosolic DNA aggregates virtually indistinguishable from micronuclei. RNase H2-dependent micronuclei were surrounded by nuclear lamina and most of them contained damaged DNA. Importantly, they induced expression of interferon-stimulated genes (ISGs) and co-localized with the nucleic acid sensor cGAS. Moreover, micronuclei associated with RNase H2 deficiency were cleared by autophagy. Consequently, induction of autophagy by pharmacological mTOR inhibition resulted in a significant reduction of cytosolic DNA and the accompanied interferon signature. Autophagy induction might therefore represent a viable therapeutic option for RNase H2-dependent disease. Endogenous retroelements have previously been proposed as a source of self-nucleic acids triggering inappropriate activation of the immune system in AGS. We used human RNase H2-knockout cells generated by CRISPR/Cas9 to investigate the impact of RNase H2 on retroelement propagation. Surprisingly, replication of LINE-1 and Alu elements was blunted in cells lacking RNase H2, establishing RNase H2 as essential host factor for the mobilisation of endogenous retrotransposons.

Trans-ethnic meta-regression of genome-wide association studies accounting for ancestry increases power for discovery and improves fine-mapping resolution.

Abstract: Trans-ethnic meta-analysis of genome-wide association studies (GWAS) across diverse populations can increase power to detect complex trait loci when the underlying causal variants are shared between ancestry groups. However, heterogeneity in allelic effects between GWAS at these loci can occur that is correlated with ancestry. Here, a novel approach is presented to detect SNP association and quantify the extent of heterogeneity in allelic effects that is correlated with ancestry. We employ trans-ethnic meta-regression to model allelic effects as a function of axes of genetic variation, derived from a matrix of mean pairwise allele frequency differences between GWAS, and implemented in the MR-MEGA software. Through detailed simulations, we demonstrate increased power to detect association for MR-MEGA over fixed- and random-effects meta-analysis across a range of scenarios of heterogeneity in allelic effects between ethnic groups. We also demonstrate improved fine-mapping resolution, in loci containing a single causal variant, compared to these meta-analysis approaches and PAINTOR, and equivalent performance to MANTRA at reduced computational cost. Application of MR-MEGA to trans-ethnic GWAS of kidney function in 71,461 individuals indicates stronger signals of association than fixed-effects meta-analysis when heterogeneity in allelic effects is correlated with ancestry. Application of MR-MEGA to fine-mapping four type 2 diabetes susceptibility loci in 22,086 cases and 42,539 controls highlights: (i) strong evidence for heterogeneity in allelic effects that is correlated with ancestry only at the index SNP for the association signal at the CDKAL1 locus; and (ii) 99% credible sets with six or fewer variants for five distinct association signals.

New insights into the genetics of primary open-angle glaucoma based on meta-analyses of intraocular pressure and optic disc characteristics

Abstract: Primary open-angle glaucoma (POAG), the most common optic neuropathy, is a heritable disease. Siblings of POAG cases have a ten-fold increased risk of developing the disease. Intraocular pressure (IOP) and optic nerve head characteristics are used clinically to predict POAG risk. We conducted a genome-wide association meta-analysis of IOP and optic disc parameters and validated our findings in multiple sets of POAG cases and controls. Using imputation to the 1000 genomes (1000G) reference set, we identified 9 new genomic regions associated with vertical cup-disc ratio (VCDR) and 1 new region associated with IOP. Additionally, we found 5 novel loci for optic nerve cup area and 6 for disc area. Previously it was assumed that genetic variation influenced POAG either through IOP or via changes to the optic nerve head; here we present evidence that some genomic regions affect both IOP and the disc parameters. We characterized the effect of the novel loci through pathway analysis and found that pathways involved are not entirely distinct as assumed so far. Further, we identified a novel association between CDKN1A and POAG. Using a zebrafish model we show that six6b (associated with POAG and optic nerve head variation) alters the expression of cdkn1a. In summary, we have identified several novel genes influencing the major clinical risk predictors of POAG and showed that genetic variation in CDKN1A is important in POAG risk.

Conditional eQTL analysis reveals allelic heterogeneity of gene expression

Abstract: In recent years, multiple eQTL (expression quantitative trait loci) catalogs have become available that can help understand the functionality of complex trait-related single nucleotide polymorphisms (SNPs). In eQTL catalogs, gene expression is often strongly associated with multiple SNPs, which may reflect either one or multiple independent associations. Conditional eQTL analysis allows a distinction between dependent and independent eQTLs. We performed conditional eQTL analysis in 4,896 peripheral blood microarray gene expression samples. Our analysis showed that 35% of genes with a cis eQTL have at least two independent cis eQTLs; for several genes up to 13 independent cis eQTLs were identified. Also, 12% (671) of the independent cis eQTLs identified in conditional analyses were not significant in unconditional analyses. The number of GWAS catalog SNPs identified as eQTL in the conditional analyses increases with 24% as compared to unconditional analyses. We provide an online conditional cis eQTL mapping catalog for whole blood (https://eqtl.onderzoek.io/), which can be used to lookup eQTLs more accurately than in standard unconditional whole blood eQTL databases.

Progranulin regulates lysosomal function and biogenesis through acidification of lysosomes.

Abstract: Progranulin (PGRN) haploinsufficiency resulting from loss-of-function mutations in the PGRN gene causes frontotemporal lobar degeneration accompanied by TDP-43 accumulation, and patients with homozygous mutations in the PGRN gene present with neuronal ceroid lipofuscinosis. Although it remains unknown why PGRN deficiency causes neurodegenerative diseases, there is increasing evidence that PGRN is implicated in lysosomal functions. Here, we show PGRN is a secretory lysosomal protein that regulates lysosomal function and biogenesis by controlling the acidification of lysosomes. PGRN gene expression and protein levels increased concomitantly with the increase of lysosomal biogenesis induced by lysosome alkalizers or serum starvation. Down-regulation or insufficiency of PGRN led to the increased lysosomal gene expression and protein levels, while PGRN overexpression led to the decreased lysosomal gene expression and protein levels. In particular, the level of mature cathepsin D (CTSDmat) dramatically changed depending upon PGRN levels. The acidification of lysosomes was facilitated in cells transfected with PGRN. Then, this caused degradation of CTSDmat by cathepsin B. Secreted PGRN is incorporated into cells via sortilin or cation-independent mannose 6-phosphate receptor, and facilitated the acidification of lysosomes and degradation of CTSDmat. Moreover, the change of PGRN levels led to a cell-type-specific increase of insoluble TDP-43. In the brain tissue of FTLD-TDP patients with PGRN deficiency, CTSD and phosphorylated TDP-43 accumulated in neurons. Our study provides new insights into the physiological function of PGRN and the role of PGRN insufficiency in the pathogenesis of neurodegenerative diseases.

Emerging mechanisms of aminoacyl-tRNA synthetase mutations in recessive and dominant human disease.

Abstract: Aminoacyl-tRNA synthetases (ARSs) are responsible for charging amino acids to cognate tRNA molecules, which is the essential first step of protein translation. Interestingly, mutations in genes encoding ARS enzymes have been implicated in a broad spectrum of human inherited diseases. Bi-allelic mutations in ARSs typically cause severe, early-onset, recessive diseases that affect a wide range of tissues. The vast majority of these mutations show loss-of-function effects and impair protein translation. However, it is not clear how a subset cause tissue-specific phenotypes. In contrast, dominant ARS-mediated diseases specifically affect the peripheral nervous system-most commonly causing axonal peripheral neuropathy-and usually manifest later in life. These neuropathies are linked to heterozygosity for missense mutations in five ARS genes, which points to a shared mechanism of disease. However, it is not clear if a loss-of-function mechanism or a toxic gain-of-function mechanism is responsible for ARS-mediated neuropathy, or if a combination of these mechanisms operate on a mutation-specific basis. Here, we review our current understanding of recessive and dominant ARS-mediated disease. We also propose future directions for defining the molecular mechanisms of ARS mutations toward designing therapies for affected patient populations.

Inhibition of mitochondrial fragmentation protects against Alzheimer's disease in rodent model.

Abstract: Mitochondrial dysfunction is an early prominent feature in susceptible neurons in the brain of patients with Alzheimer's disease, which likely plays a critical role in the pathogenesis of disease. Increasing evidence suggests abnormal mitochondrial dynamics as important underlying mechanisms. In this study, we characterized marked mitochondrial fragmentation and abnormal mitochondrial distribution in the pyramidal neurons along with mitochondrial dysfunction in the brain of Alzheimer's disease mouse model CRND8 as early as 3 months of age before the accumulation of amyloid pathology. To establish the pathogenic significance of these abnormalities, we inhibited mitochondrial fragmentation by the treatment of mitochondrial division inhibitor 1 (mdivi-1), a mitochondrial fission inhibitor. Mdivi-1 treatment could rescue both mitochondrial fragmentation and distribution deficits and improve mitochondrial function in the CRND8 neurons both in vitro and in vivo. More importantly, the amelioration of mitochondrial dynamic deficits by mdivi-1 treatment markedly decreased extracellular amyloid deposition and Aβ1-42/Aβ1-40 ratio, prevented the development of cognitive deficits in Y-maze test and improved synaptic parameters. Our findings support the notion that abnormal mitochondrial dynamics plays an early and causal role in mitochondrial dysfunction and Alzheimer's disease-related pathological and cognitive impairments in vivo and indicate the potential value of restoration of mitochondrial dynamics as an innovative therapeutic strategy for Alzheimer's disease.

MicroRNA-455-3p as a potential peripheral biomarker for Alzheimer's disease.

Abstract: The purpose of our study was to identify microRNAs (miRNAs) as early detectable peripheral biomarkers in Alzheimer’s disease (AD). To achieve our objective, we assessed miRNAs in serum samples from AD patients and Mild cognitive impairment (MCI) subjects relative to healthy controls. We used Affymetrix microarray analysis and validated differentially expressed miRNAs using qRT-PCR. We further validated miRNA data using AD postmortem brains, amyloid precursor protein transgenic mice and AD cell lines. We identified a gradual upregulation of four miRNAs: miR-455-3p, miR-4668-5p, miR-3613-3p and miR-4674. A fifth miRNA, mir-6722, was down-regulated in persons with AD and mild cognitive impairment compared with controls. Validation analysis by qRT-PCR showed significant upregulation of only miR-455-3p (P = 0.007) and miR-4668-5p (P = 0.016) in AD patients compared with healthy controls. Furthermore, qRT-PCR analysis of the AD postmortem brains with different Braak stages also showed upregulation of miR-455-3p (P = 0.016). However, receiver operating characteristic curves (ROC) curve analysis revealed a significant area under curve (AUC) value only for miR-455-3p in the serum (AUROC = 0.79; P = 0.015) and brains (AUROC = 0.86; P = 0.016) of AD patients. Expression analysis of amyloid precursor protein transgenic mice also revealed high level of mmu-miR-455-3p (P = 0.004) in the cerebral cortex (AD-affected) region of brain and low in the non-affected area, i.e. cerebellum. Furthermore, human and mouse neuroblastoma cells treated with the amyloid-β(1–42) peptide also showed a similarly higher expression of miR-455-3p. Functional analysis of differentially expressed miRNAs via the miR-path indicated that miR-455-3p was associated in the regulation of several biological pathways. Genes associated with these pathways were found to have a crucial role in AD pathogenesis. An increase in miR-455-3p expression found in AD patients and Aβ pathologies unveiled its biomarker characteristics and a precise role in AD pathogenesis.

Genetics of age-related macular degeneration (AMD).

Abstract: Age-related macular degeneration (AMD) is a progressive blinding disease and represents the leading cause of visual impairment in the aging population. AMD affects central vision which impairs one's ability to drive, read and recognize faces. There is no cure for this disease and current treatment modalities for the exudative form of the disease require repeated intravitreal injections which may be painful, are incompletely efficacious, and represent a significant treatment burden for both the patient and physician. As such, AMD represents a significant and important clinical problem.It is anticipated that in three years' time, 196 million individuals will be affected with AMD. Over 250 billion dollars per year are spent on care for AMD patients in the US. Over half of the heritability is explained by two major loci, thus AMD is considered the most well genetically defined of the complex disorders. A recent GWAS on 43,566 subjects identified novel loci and pathways associated with AMD risk, which has provided an excellent platform for additional functional studies. Genetic variants have been investigated, particularly with respect to anti-VEGF treatment, however to date, no pharmacogenomic associations have been consistently identified across these studies. It may be that if the goal of personalized medicine is to be realized and biomarkers are to have predictive value for determining the magnitude of risk for AMD at the genetic level, one will need to examine the relationships between these pathways across disease state and relative to modifiable risk factors such as hypertension, smoking, body mass index, and hypercholesterolemia. Further studies investigating protective alleles in populations with low AMD prevalence may lead to this goal.

Sodium butyrate rescues dopaminergic cells from alpha-synuclein-induced transcriptional deregulation and DNA damage.

Abstract: Alpha-synuclein (aSyn) is considered a major culprit in Parkinson's disease (PD) pathophysiology. However, the precise molecular function of the protein remains elusive. Recent evidence suggests that aSyn may play a role on transcription regulation, possibly by modulating the acetylation status of histones. Our study aimed at evaluating the impact of wild-type (WT) and mutant A30P aSyn on gene expression, in a dopaminergic neuronal cell model, and decipher potential mechanisms underlying aSyn-mediated transcriptional deregulation. We performed gene expression analysis using RNA-sequencing in Lund Human Mesencephalic (LUHMES) cells expressing endogenous (control) or increased levels of WT or A30P aSyn. Compared to control cells, cells expressing both aSyn variants exhibited robust changes in the expression of several genes, including downregulation of major genes involved in DNA repair. WT aSyn, unlike A30P aSyn, promoted DNA damage and increased levels of phosphorylated p53. In dopaminergic neuronal cells, increased aSyn expression led to reduced levels of acetylated histone 3. Importantly, treatment with sodium butyrate, a histone deacetylase inhibitor (HDACi), rescued WT aSyn-induced DNA damage, possibly via upregulation of genes involved in DNA repair. Overall, our findings provide novel and compelling insight into the mechanisms associated with aSyn neurotoxicity in dopaminergic cells, which could be ameliorated with an HDACi. Future studies will be crucial to further validate these findings and to define novel possible targets for intervention in PD.

Alpha-synuclein induces the unfolded protein response in Parkinson’s disease SNCA triplication iPSC-derived neurons

Abstract: The recent generation of induced pluripotent stem cells (iPSCs) from a patient with Parkinson's disease (PD) resulting from triplication of the α-synuclein (SNCA) gene locus allows unprecedented opportunities to explore its contribution to the molecular pathogenesis of PD. We used the double-nicking CRISPR/Cas9 system to conduct site-specific mutagenesis of SNCA in these cells, generating an isogenic iPSC line with normalized SNCA gene dosage. Comparative gene expression analysis of neuronal derivatives from these iPSCs revealed an ER stress phenotype, marked by induction of the IRE1α/XBP1 axis of the unfolded protein response (UPR) and culminating in terminal UPR activation. Neuropathological analysis of post-mortem brain tissue demonstrated that pIRE1α is expressed in PD brains within neurons containing elevated levels of α-synuclein or Lewy bodies. Having used this pair of isogenic iPSCs to define this phenotype, these cells can be further applied in UPR-targeted drug discovery towards the development of disease-modifying therapeutics.

The DNA damage response (DDR) is induced by the C9orf72 repeat expansion in amyotrophic lateral sclerosis.

Abstract: Amyotrophic lateral sclerosis (ALS) is a rapidly progressing neurodegenerative disease affecting motor neurons. Hexanucleotide (GGGGCC) repeat expansions in a non-coding region of C9orf72 are the major cause of familial ALS and frontotemporal dementia (FTD) worldwide. The C9orf72 repeat expansion undergoes repeat-associated non-ATG (RAN) translation to produce five dipeptide repeat proteins (DRPs), including poly(GR) and poly(PR). Whilst it remains unclear how mutations in C9orf72 lead to neurodegeneration in ALS/FTD, dysfunction to the nucleolus and R loop formation are implicated as pathogenic mechanisms. These events can damage DNA and hence genome integrity. Cells activate the DNA damage response (DDR) with the aim of repairing this damage. However, if the damage cannot be repaired, apoptosis is triggered. In lumbar motor neurons from C9orf72-positive ALS patients, we demonstrate significant up-regulation of markers of the DDR compared to controls: phosphorylated histone 2AX (γ-H2AX), phosphorylated ataxia telangiectasia mutated (p-ATM), cleaved poly (ADP-Ribose) polymerase 1 (PARP-1) and tumour suppressor p53-binding protein (53BP1). Similarly, significant up-regulation of γ-H2AX and p-ATM was detected in neuronal cells expressing poly(GR)100 and poly(PR)100 compared to controls, revealing that DNA damage is triggered by the DRPs. Nucleophosmin (NPM1) is a histone chaperone induced during the DDR, which interacts with APE1 to enhance DNA repair. We also demonstrate that more NPM1 precipitates with APE1 in C9orf72 patients compared to controls. Furthermore, overexpression of NPM1 inhibits apoptosis in cells expressing poly(GR)100 and poly(PR)100. This study therefore demonstrates that DNA damage is activated by the C9orf72 repeat expansion in ALS.

Mitochondrial dysfunction underlies cognitive defects as a result of neural stem cell depletion and impaired neurogenesis.

Abstract: Mitochondrial dysfunction is a common feature of many genetic disorders that target the brain and cognition. However, the exact role these organelles play in the etiology of such disorders is not understood. Here, we show that mitochondrial dysfunction impairs brain development, depletes the adult neural stem cell (NSC) pool and impacts embryonic and adult neurogenesis. Using deletion of the mitochondrial oxidoreductase AIF as a genetic model of mitochondrial and neurodegenerative diseases revealed the importance of mitochondria in multiple steps of the neurogenic process. Developmentally, impaired mitochondrial function causes defects in NSC self-renewal, neural progenitor cell proliferation and cell cycle exit, as well as neuronal differentiation. Sustained mitochondrial dysfunction into adulthood leads to NSC depletion, loss of adult neurogenesis and manifests as a decline in brain function and cognitive impairment. These data demonstrate that mitochondrial dysfunction, as observed in genetic mitochondrial and neurodegenerative diseases, underlies the decline of brain function and cognition due to impaired stem cell maintenance and neurogenesis.

Progranulin functions as a cathepsin D chaperone to stimulate axonal outgrowth in vivo

Abstract: Loss of function mutations in progranulin (GRN) cause frontotemporal dementia, but how GRN haploinsufficiency causes neuronal dysfunction remains unclear. We previously showed that GRN is neurotrophic in vitro. Here, we used an in vivo axonal outgrowth system and observed a delayed recovery in GRN-/- mice after facial nerve injury. This deficit was rescued by reintroduction of human GRN and relied on its C-terminus and on neuronal GRN production. Transcriptome analysis of the facial motor nucleus post injury identified cathepsin D (CTSD) as the most upregulated gene. In aged GRN-/- cortices, CTSD was also upregulated, but the relative CTSD activity was reduced and improved upon exogenous GRN addition. Moreover, GRN and its C-terminal granulin domain granulinE (GrnE) both stimulated the proteolytic activity of CTSD in vitro. Pull-down experiments confirmed a direct interaction between GRN and CTSD. This interaction was also observed with GrnE and stabilized the CTSD enzyme at different temperatures. Investigating the importance of this interaction for axonal regeneration in vivo we found that, although individually tolerated, a combined reduction of GRN and CTSD synergistically reduced axonal outgrowth. Our data links the neurotrophic effect of GRN and GrnE with a lysosomal chaperone function on CTSD to maintain its proteolytic capacity.

A frequent oligogenic involvement in congenital hypothyroidism

Abstract: Congenital hypothyroidism (CH), the most frequent form of preventable mental retardation, is predicted to have a relevant genetic origin. However, CH is frequently reported to be sporadic and candidate gene variations were found in <10% of the investigated patients. Here, we characterize the involvement of 11 candidate genes through a systematic Next Generation Sequencing (NGS) analysis. The NGS was performed in 177 unrelated CH patients (94 gland-in-situ; 83 dysgenesis) and in 3,538 control subjects. Non-synonymous or splicing rare variants (MAF < 0.01) were accepted, and their functional impact was predicted by a comprehensive bioinformatic approach and co-segregation studies. The frequency of variations in cases and controls was extended to 18 CH-unrelated genes. At least one rare variant was accepted in 103/177 patients. Monogenic recessive forms of the disease were found in five cases, but oligogenic involvement was detected in 39 patients. The 167 variations were found to affect all genes independently of the CH phenotype. These findings were replicated in an independent cohort of additional 145 CH cases. When compared to 3,538 controls, the CH population was significantly enriched with disrupting variants in the candidate genes (P = 5.5 × 10-7), but not with rare variations in CH-unrelated genes. Co-segregation studies of the hypothyroid phenotype with multiple gene variants in several pedigrees confirmed the potential oligogenic origin of CH. The systematic NGS approach reveals the frequent combination of rare variations in morphogenetic or functional candidate genes in CH patients independently of phenotype. The oligogenic origin represents a suitable explanation for the frequent sporadic CH occurrence.

Circular RNA profiling reveals that circular RNAs from ANXA2 can be used as new biomarkers for multiple sclerosis.

Abstract: Multiple sclerosis is an autoimmune disease, with higher prevalence in women, in whom the immune system is dysregulated. This dysregulation has been shown to correlate with changes in transcriptome expression as well as in gene-expression regulators, such as non-coding RNAs (e.g. microRNAs). Indeed, some of these have been suggested as biomarkers for multiple sclerosis even though few biomarkers have reached the clinical practice. Recently, a novel family of non-coding RNAs, circular RNAs, has emerged as a new player in the complex network of gene-expression regulation. MicroRNA regulation function through a 'sponge system' and a RNA splicing regulation function have been proposed for the circular RNAs. This regulating role together with their high stability in biofluids makes them seemingly good candidates as biomarkers. Given the dysregulation of both protein-coding and non-coding transcriptome that have been reported in multiple sclerosis patients, we hypothesised that circular RNA expression may also be altered. Therefore, we carried out expression profiling of 13.617 circular RNAs in peripheral blood leucocytes from multiple sclerosis patients and healthy controls finding 406 differentially expressed (P-value 1.5) and demonstrate after validation that, circ_0005402 and circ_0035560 are underexpressed in multiple sclerosis patients and could be used as biomarkers of the disease.

Repetitive element transcripts are elevated in the brain of C9orf72 ALS/FTLD patients.

Abstract: Significant transcriptome alterations are detected in the brain of patients with amyotrophic lateral sclerosis (ALS), including carriers of the C9orf72 repeat expansion and C9orf72-negative sporadic cases. Recently, the expression of repetitive element transcripts has been associated with toxicity and, while increased repetitive element expression has been observed in several neurodegenerative diseases, little is known about their contribution to ALS. To assess whether aberrant expression of repetitive element sequences are observed in ALS, we analysed RNA sequencing data from C9orf72-positive and sporadic ALS cases, as well as healthy controls. Transcripts from multiple classes and subclasses of repetitive elements (LINEs, endogenous retroviruses, DNA transposons, simple repeats, etc.) were significantly increased in the frontal cortex of C9orf72 ALS patients. A large collection of patient samples, representing both C9orf72 positive and negative ALS, ALS/FTLD, and FTLD cases, was used to validate the levels of several repetitive element transcripts. These analyses confirmed that repetitive element expression was significantly increased in C9orf72-positive compared to C9orf72-negative or control cases. While previous studies suggest an important link between TDP-43 and repetitive element biology, our data indicate that TDP-43 pathology alone is insufficient to account for the observed changes in repetitive elements in ALS/FTLD. Instead, we found that repetitive element expression positively correlated with RNA polymerase II activity in postmortem brain, and pharmacologic modulation of RNA polymerase II activity altered repetitive element expression in vitro. We conclude that increased RNA polymerase II activity in ALS/FTLD may lead to increased repetitive element transcript expression, a novel pathological feature of ALS/FTLD.

Progranulin-mediated deficiency of cathepsin D results in FTD and NCL-like phenotypes in neurons derived from FTD patients.

Abstract: Frontotemporal dementia (FTD) encompasses a group of neurodegenerative disorders characterized by cognitive and behavioral impairments. Heterozygous mutations in progranulin (PGRN) cause familial FTD and result in decreased PGRN expression, while homozygous mutations result in complete loss of PGRN expression and lead to the neurodegenerative lysosomal storage disorder neuronal ceroid lipofuscinosis (NCL). However, how dose-dependent PGRN mutations contribute to these two different diseases is not well understood. Using iPSC-derived human cortical neurons from FTD patients harboring PGRN mutations, we demonstrate that PGRN mutant neurons exhibit decreased nuclear TDP-43 and increased insoluble TDP-43, as well as enlarged electron-dense vesicles, lipofuscin accumulation, fingerprint-like profiles and granular osmiophilic deposits, suggesting that both FTD and NCL-like pathology are present in PGRN patient neurons as compared to isogenic controls. PGRN mutant neurons also show impaired lysosomal proteolysis and decreased activity of the lysosomal enzyme cathepsin D. Furthermore, we find that PGRN interacts with cathepsin D, and that PGRN increases the activity of cathepsin D but not cathepsins B or L. Finally, we show that granulin E, a cleavage product of PGRN, is sufficient to increase cathepsin D activity. This functional relationship between PGRN and cathepsin D provides a possible explanation for overlapping NCL-like pathology observed in patients with mutations in PGRN or CTSD, the gene encoding cathepsin D. Together, our work identifies PGRN as an activator of lysosomal cathepsin D activity, and suggests that decreased cathepsin D activity due to loss of PGRN contributes to both FTD and NCL pathology in a dose-dependent manner.

Vps35 in cooperation with LRRK2 regulates synaptic vesicle endocytosis through the endosomal pathway in Drosophila.

Abstract: Mutations of the retromer component Vps35 and endosomal kinase LRRK2 are linked to autosomal dominant forms of familial Parkinson's disease (PD). However, the physiological and pathological roles of Vps35 and LRRK2 in neuronal functions are poorly understood. Here, we demonstrated that the loss of Drosophila Vps35 (dVps35) affects synaptic vesicle recycling, dopaminergic synaptic release and sleep behavior associated with dopaminergic activity, which is rescued by the expression of wild-type dVps35 but not the PD-associated mutant dVps35 D647N. Drosophila LRRK2 dLRRK together with Rab5 and Rab11 is also implicated in synaptic vesicle recycling, and the manipulation of these activities improves the Vps35 synaptic phenotypes. These findings indicate that defects of synaptic vesicle recycling in which two late-onset PD genes, Vps35 and LRRK2, are involved could be key aspects of PD etiology.

Imputation of Orofacial Clefting Data Identifies Novel Risk Loci and Sheds Light on the Genetic Background of Cleft Lip ± Cleft Palate and Cleft Palate Only.

Abstract: Nonsyndromic cleft lip with or without cleft palate (nsCL/P) is among the most common human birth defects with multifactorial etiology. Here, we present results from a genome-wide imputation study of nsCL/P in which, after adding replication cohort data, four novel risk loci for nsCL/P are identified (at chromosomal regions 2p21, 14q22, 15q24 and 19p13). On a systematic level, we show that the association signals within this high-density dataset are enriched in functionally-relevant genomic regions that are active in both human neural crest cells (hNCC) and mouse embryonic craniofacial tissue. This enrichment is also detectable in hNCC regions primed for later activity. Using GCTA analyses, we suggest that 30% of the estimated variance in risk for nsCL/P in the European population can be attributed to common variants, with 25.5% contributed to by the 24 risk loci known to date. For each of these, we identify credible SNPs using a Bayesian refinement approach, with two loci harbouring only one probable causal variant. Finally, we demonstrate that there is no polygenic component of nsCL/P detectable that is shared with nonsyndromic cleft palate only (nsCPO). Our data suggest that, while common variants are strongly contributing to risk for nsCL/P, they do not seem to be involved in nsCPO which might be more often caused by rare deleterious variants. Our study generates novel insights into both nsCL/P and nsCPO etiology and provides a systematic framework for research into craniofacial development and malformation.

Epigenome-wide analysis of DNA methylation in lung tissue shows concordance with blood studies and identifies tobacco smoke-inducible enhancers.

Abstract: Smoking-associated DNA hypomethylation has been observed in blood cells and linked to lung cancer risk. However, its cause and mechanistic relationship to lung cancer remain unclear. We studied the association between tobacco smoking and epigenome-wide methylation in non-tumor lung (NTL) tissue from 237 lung cancer cases in the Environment And Genetics in Lung cancer Etiology study, using the Infinium HumanMethylation450 BeadChip. We identified seven smoking-associated hypomethylated CpGs (P < 1.0 × 10-7), which were replicated in NTL data from The Cancer Genome Atlas. Five of these loci were previously reported as hypomethylated in smokers' blood, suggesting that blood-based biomarkers can reflect changes in the target tissue for these loci. Four CpGs border sequences carrying aryl hydrocarbon receptor binding sites and enhancer-specific histone modifications in primary alveolar epithelium and A549 lung adenocarcinoma cells. A549 cell exposure to cigarette smoke condensate increased these enhancer marks significantly and stimulated expression of predicted target xenobiotic response-related genes AHRR (P = 1.13 × 10-62) and CYP1B1 (P < 2.49 × 10-61). Expression of both genes was linked to smoking-related transversion mutations in lung tumors. Thus, smoking-associated hypomethylation may be a consequence of enhancer activation, revealing environmentally-induced regulatory elements implicated in lung carcinogenesis.

Novel nesprin-1 mutations associated with dilated cardiomyopathy cause nuclear envelope disruption and defects in myogenesis.

Abstract: Nesprins-1 and -2 are highly expressed in skeletal and cardiac muscle and together with SUN (Sad1p/UNC84)-domain containing proteins and lamin A/C form the LInker of Nucleoskeleton-and-Cytoskeleton (LINC) bridging complex at the nuclear envelope (NE). Mutations in nesprin-1/2 have previously been found in patients with autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) as well as dilated cardiomyopathy (DCM). In this study, three novel rare variants (R8272Q, S8381C and N8406K) in the C-terminus of the SYNE1 gene (nesprin-1) were identified in seven DCM patients by mutation screening. Expression of these mutants caused nuclear morphology defects and reduced lamin A/C and SUN2 staining at the NE. GST pull-down indicated that nesprin-1/lamin/SUN interactions were disrupted. Nesprin-1 mutations were also associated with augmented activation of the ERK pathway in vitro and in hearts in vivo. During C2C12 muscle cell differentiation, nesprin-1 levels are increased concomitantly with kinesin light chain (KLC-1/2) and immunoprecipitation and GST pull-down showed that these proteins interacted via a recently identified LEWD domain in the C-terminus of nesprin-1. Expression of nesprin-1 mutants in C2C12 cells caused defects in myoblast differentiation and fusion associated with dysregulation of myogenic transcription factors and disruption of the nesprin-1 and KLC-1/2 interaction at the outer nuclear membrane. Expression of nesprin-1α2 WT and mutants in zebrafish embryos caused heart developmental defects that varied in severity. These findings support a role for nesprin-1 in myogenesis and muscle disease, and uncover a novel mechanism whereby disruption of the LINC complex may contribute to the pathogenesis of DCM.

Dimethyl fumarate mediates Nrf2-dependent mitochondrial biogenesis in mice and humans

Abstract: The induction of mitochondrial biogenesis could potentially alleviate mitochondrial and muscle disease. We show here that dimethyl fumarate (DMF) dose-dependently induces mitochondrial biogenesis and function dosed to cells in vitro, and also dosed in vivo to mice and humans. The induction of mitochondrial gene expression is more dependent on DMF's target Nrf2 than hydroxycarboxylic acid receptor 2 (HCAR2). Thus, DMF induces mitochondrial biogenesis primarily through its action on Nrf2, and is the first drug demonstrated to increase mitochondrial biogenesis with in vivo human dosing. This is the first demonstration that mitochondrial biogenesis is deficient in Multiple Sclerosis patients, which could have implications for MS pathophysiology and therapy. The observation that DMF stimulates mitochondrial biogenesis, gene expression and function suggests that it could be considered for mitochondrial disease therapy and/or therapy in muscle disease in which mitochondrial function is important.