ABSTRACT
BACKGROUND: Anorexia nervosa (AN) is a severe psychiatric disorder associated with frequent relapses and variability in treatment responses. Previous literature suggested that such variability is influenced by premorbid vulnerabilities such as abnormalities of the reward system. Several factors may indicate these vulnerabilities, such as neurocognitive markers (tendency to favour delayed reward, poor cognitive flexibility, abnormal decision process), genetic and epigenetic markers, biological and hormonal markers, and physiological markers.The present study will aim to identify markers that can predict body mass index (BMI) stability 6 months after discharge. The secondary aim of this study will be focused on characterising the biological, genetic, epigenetic and neurocognitive markers of remission in AN. METHODS AND ANALYSIS: One hundred and twenty-five (n=125) female adult inpatients diagnosed with AN will be recruited and evaluated at three different times: at the beginning of hospitalisation, when discharged and 6 months later. Depending on the BMI at the third visit, patients will be split into two groups: stable remission (BMI≥18.5 kg/m²) or unstable remission (BMI<18.5 kg/m²). One hundred (n=100) volunteers will be included as healthy controls.Each visit will consist in self-reported inventories (measuring depression, anxiety, suicidal thoughts and feelings, eating disorders symptoms, exercise addiction and the presence of comorbidities), neurocognitive tasks (Delay Discounting Task, Trail-Making Test, Brixton Test and Slip-of-action Task), the collection of blood samples, the repeated collection of blood samples around a standard meal and MRI scans at rest and while resolving a delay discounting task.Analyses will mainly consist in comparing patients stabilised 6 months later and patients who relapsed during these 6 months. ETHICS AND DISSEMINATION: Investigators will ask all participants to give written informed consent prior to participation, and all data will be recorded anonymously. The study will be conducted according to ethics recommendations from the Helsinki declaration (World Medical Association, 2013). It was registered on clinicaltrials.gov on 25 August 2020 as 'Remission Factors in Anorexia Nervosa (REMANO)', with the identifier NCT04560517 (for more details, see https://clinicaltrials.gov/ct2/show/record/NCT04560517). The present article is based on the latest protocol version from 29 November 2019. The sponsor, Institut National de la Santé Et de la Recherche Médicale (INSERM, https://www.inserm.fr/), is an academic institution responsible for the monitoring of the study, with an audit planned on a yearly basis.The results will be published after final analysis in the form of scientific articles in peer-reviewed journals and may be presented at national and international conferences. TRIAL REGISTRATION NUMBER: clinicaltrials.govNCT04560517.
Subject(s)
Anorexia Nervosa , Biomarkers , Body Mass Index , Humans , Anorexia Nervosa/genetics , Anorexia Nervosa/therapy , Anorexia Nervosa/blood , Female , Prospective Studies , Biomarkers/blood , Adult , Case-Control Studies , France , Neuroimaging , Young Adult , Adolescent , Remission InductionABSTRACT
LEAP-2 is a ghrelin antagonist with an anorexigenic drive. This study investigates the evolution of plasma ghrelin and LEAP-2 concentrations in 29 patients with anorexia nervosa (AN) before and after refeeding and compares it to physiological adaptations during fasting in healthy controls or to mouse model of chronic food restriction and refeeding. Acute and chronic food restriction decrease LEAP-2 and increase ghrelin concentrations in both humans and mice, while patients with AN displayed higher ghrelin and LEAP-2 concentrations before than after refeeding (p = 0.043). After 6 months follow-up, patients with unstable weight gain (n = 17) had significantly decreased LEAP-2 concentrations after refeeding (p = 0.044), in contrast to patients with stable weight gain (n = 12). We provide evidence that the ghrelin/LEAP-2 system is not regulated according to the nutritional status in AN, in contrast to what is physiologically expected when coping with food restriction.
ABSTRACT
OBJECTIVES: In eating disorders, particularly anorexia nervosa (AN), patients exhibit intense physical activity which is inappropriate regarding food restriction and chronic undernutrition, and exacerbates weight loss and energy deprivation. Rodent models of food restriction exhibit increased running wheel activity in the food anticipation period, also known as Food Anticipatory Activity (FAA). FAA probably has various physiological and/or neurobiological origins. Plasma concentrations of the orexigenic hormone ghrelin are, for example, increased during FAA. We hypothesize that the drive for physical activity in chronic food restriction is triggered by metabolic factors but also relies on motivational aspects that we aim to decipher in this study. METHODS: Young female C57Bl6/J mice were exposed to a paradigm based on a progressive 50% quantitative food restriction alone (FR) or associated with running wheel activity (Food Restriction Wheel: FRW) in their home-cage during 15 days. We measured preference for running wheel in a three-chamber apparatus in which animals could choose to explore either a known running wheel or a novel object. Testing took place either during resting or during FAA. We calculated the time spent in each compartment and the activity in running wheels. After progressive refeeding over 10 days, mice were tested again when refed. Plasma levels of both ghrelin isoforms were measured with selective immunoassays. RESULTS: When tested during FAA period, food restricted mice displayed increased preference for the running wheel compared to ad libitum fed controls. Both FR and FRW mice exhibited increased running time and distance in the wheel and running distance was correlated with ghrelin levels. Similar preference and behavior were found when testing took place during the resting period. Animals housed without an active wheel also exhibited active running. Progressive refeeding resulted in body weight restoration, a decrease in FAA and completely abolished preference for the running wheel. Refed animals displayed similar behavior as ad libitum fed controls. CONCLUSIONS: These data provide evidence that food restriction-induced physical activity is closely correlated with metabolic adaptations to nutritional status implicating ghrelin in the quantity of physical activity.
Subject(s)
Eating , Ghrelin , Mice , Female , Animals , Eating/physiology , Ghrelin/metabolism , Motor Activity/physiology , Body Weight/physiology , FoodABSTRACT
Oligophrenin-1 (OPHN1) is a Rho-GTPase-activating protein (RhoGAP), whose mutations are associated with X-linked intellectual disability (XLID). OPHN1 is enriched at the synapse in both pre- and postsynaptic compartments, where it regulates the RhoA/ROCK/MLC2 signaling pathway, playing a critical role in cytoskeleton remodeling and vesicle recycling. Ophn1 knockout (KO) adult mice display some behavioral deficits in multiple tasks, reminiscent of some symptoms in the human pathology. We also previously reported a reduction in dendritic spine density in the adult hippocampus of KO mice. Yet the nature of the deficits occurring in these mice during postnatal development remains elusive. Here, we show that juvenile KO mice present normal basal synaptic transmission, but altered synaptic plasticity, with a selective impairment in long-term depression, but no change in long-term potentiation. This contrasts with the functional deficits that these mice display at the adult stage, as we found that both basal synaptic transmission and long-term potentiation are reduced at later stages, due to presynaptic alterations. In addition, the number of excitatory synapses in adult is increased, suggesting some unsuccessful compensation. Altogether, these results suggest that OPHN1 function at synapses is differentially affected during maturation of the brain, which provides some therapeutic opportunities for early intervention.
Subject(s)
Cytoskeletal Proteins , GTPase-Activating Proteins , Hippocampus , Intellectual Disability , Synaptic Transmission , Animals , Cytoskeletal Proteins/genetics , Cytoskeletal Proteins/metabolism , GTPase-Activating Proteins/genetics , GTPase-Activating Proteins/metabolism , Hippocampus/metabolism , Intellectual Disability/genetics , Intellectual Disability/pathology , Mice , Mice, Knockout , Nuclear Proteins/genetics , Nuclear Proteins/metabolismABSTRACT
INTRODUCTION: While the vast majority of research investigating the role of ghrelin or its receptor, GHS-R1a, in growth, feeding, and metabolism has been conducted in male rodents, very little is known about sex differences in this system. Furthermore, the role of GHS-R1a signaling in the control of pulsatile GH secretion and its link with growth or metabolic parameters has never been characterized. METHODS: We assessed the sex-specific contribution of GHS-R1a signaling in the activity of the GH/IGF-1 axis, metabolic parameters, and feeding behavior in adolescent (5-6 weeks old) or adult (10-19 weeks old) GHS-R KO (Ghsr-/-) and WT (Ghsr+/+) male and female mice. RESULTS: Adult Ghsr-/- male and female mice displayed deficits in weight and linear growth that were correlated with reduced GH pituitary contents in males only. GHS-R1a deletion was associated with reduced meal frequency and increased meal intervals, as well as reduced hypothalamic GHRH and NPY mRNA in males, not females. In adult, GH release from Ghsr-/- mice pituitary explants ex vivo was reduced independently of the sex. However, in vivo pulsatile GH secretion decreased in adult but not adolescent Ghsr-/- females, while in males, GHS-R1a deletion was associated with reduction in pulsatile GH secretion during adolescence exclusively. In males, linear growth did not correlate with pulsatile GH secretion, but rather with ApEn, a measure that reflects irregularity of the rhythmic secretion. Fat mass, plasma leptin concentrations, or ambulatory activity did not predict differences in GH secretion. DISCUSSION/CONCLUSION: These results point to a sex-dependent dimorphic effect of GHS-R1a signaling to modulate pulsatile GH secretion and meal pattern in mice with different compensatory mechanisms occurring in the hypothalamus of adult males and females after GHS-R1a deletion. Altogether, we show that GHS-R1a signaling plays a more critical role in the regulation of pulsatile GH secretion during adolescence in males and adulthood in females.
Subject(s)
Ghrelin , Receptors, Ghrelin/metabolism , Animals , Feeding Behavior , Female , Ghrelin/metabolism , Growth Hormone/metabolism , Hypothalamus/metabolism , Male , Mice , Pituitary Gland/metabolism , Receptors, Ghrelin/geneticsABSTRACT
Using preproghrelin-deficient mice (Ghrl-/-), we previously observed that preproghrelin modulates pulsatile growth hormone (GH) secretion in post-pubertal male mice. However, the role of ghrelin and its derived peptides in the regulation of growth parameters or feeding in females is unknown. We measured pulsatile GH secretion, growth, metabolic parameters and feeding behavior in adult Ghrl-/- and Ghrl+/+ male and female mice. We also assessed GH release from pituitary explants and hypothalamic growth hormone-releasing hormone (GHRH) expression and immunoreactivity. Body weight and body fat mass, linear growth, spontaneous food intake and food intake following a 48-h fast, GH pituitary contents and GH release from pituitary explants ex vivo, fasting glucose and glucose tolerance were not different among adult Ghrl-/- and Ghrl+/+ male or female mice. In vivo, pulsatile GH secretion was decreased, while approximate entropy, that quantified orderliness of secretion, was increased in adult Ghrl-/- females only, defining more irregular GH pattern. The number of neurons immunoreactive for GHRH visualized in the hypothalamic arcuate nucleus was increased in adult Ghrl-/- females, as compared to Ghrl+/+ females, whereas the expression of GHRH was not different amongst groups. Thus, these results point to sex-specific effects of preproghrelin gene deletion on pulsatile GH secretion, but not feeding, growth or metabolic parameters, in adult mice.
Subject(s)
Ghrelin/physiology , Growth Hormone/metabolism , Pituitary Gland/metabolism , Sex Characteristics , Ultradian Rhythm , Animals , Arcuate Nucleus of Hypothalamus/cytology , Feeding Behavior , Female , Gene Deletion , Male , Mice, Inbred C57BLABSTRACT
Rett syndrome (RTT) is a rare X-linked neurodevelopmental disorder. More than 95% of classic RETT syndrome cases result from pathogenic variants in the methyl-CpG binding protein 2 (MECP2) gene. Nevertheless, it has been established that a spectrum of neuropsychiatric phenotypes is associated with MECP2 variants in both females and males. We previously reported that microtubule growth velocity and vesicle transport directionality are altered in Mecp2-deficient astrocytes from newborn Mecp2-deficient mice compared to that of their wild-type littermates suggesting deficit in microtubule dynamics. In this study, we report that administration of tubastatin A, a selective HDAC6 inhibitor, restored microtubule dynamics in Mecp2-deficient astrocytes. We furthermore report that daily doses of tubastatin A reversed early impaired exploratory behavior in male Mecp2308/y mice. These findings are a first step toward the validation of a novel treatment for RTT.
Subject(s)
Behavior, Animal , Histone Deacetylase 6/antagonists & inhibitors , Histone Deacetylase Inhibitors/therapeutic use , Methyl-CpG-Binding Protein 2/genetics , Rett Syndrome/drug therapy , Rett Syndrome/psychology , Animals , Astrocytes/metabolism , Exploratory Behavior , Female , Histone Deacetylase Inhibitors/pharmacology , Hydroxamic Acids/therapeutic use , Indoles/therapeutic use , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Microtubules/drug effects , Psychomotor Performance/drug effects , Social BehaviorABSTRACT
As a polygenic psychiatric disorder, the genetics of anorexia nervosa (AN) remains largely unexplored. Recently a large GWAS meta-analysis identified a significant SNP (rs6589488) as associated with AN. We suggested that rs6589488 might have gotten its association as being in linkage disequilibrium with unknown variants or functional intronic variants. In a selective cohort containing 51 patients diagnosed with restrictive subtype AN, we screened the whole coding region of the CADM1gene by Sanger sequencing and further investigated if these variants are associated with specific outcome. Only 13 single nucleotide polymorphisms, including 2 missense variants, 2 synonymous variants, 2 variants located at 5'-UTR and 7 intronic variants, including rs6589488, were identified in our AN cohort. The 2 missense variants, p.Val5Leu and p.Asp285Glu were not predicted to be deleterious. In conclusion, the intronic initial variant appears to be not associated with causative coding variant in the vicinity. If CADM1 is not the AN predisposition factor, the causative variant probably lies within 1 Mb of CADM1. Interestingly, among the 7 closest genes to CADM1, the nicotinamide N-methyltransferase (NNMT) gene is known to be associated with obesity. We suggest that the intronic variant in CADM1 could be in linkage disequilibrium with other causative variants located in NNMT.
Subject(s)
Anorexia Nervosa/diagnosis , Anorexia Nervosa/genetics , Cell Adhesion Molecule-1/genetics , Genetic Variation/genetics , Polymorphism, Single Nucleotide/genetics , Adult , Cohort Studies , Female , Genetic Predisposition to Disease/genetics , Genome-Wide Association Study/methods , Humans , Linkage Disequilibrium/genetics , Male , Young AdultABSTRACT
Widespread random monoallelic gene expression (RMAE) effects influence about 10% of human genes. However, the mechanisms by which RME of autosomal genes is established and those by which it is maintained both remain open questions. Because the choice of allelic expression is randomly performed cell-by-cell, the RMAE mechanism is not observable in non-clonal cell populations or in whole tissues. Several target genes of MeCP2, the gene involved in Rett syndrome (RTT), have been previously described as subject to RMAE, suggesting that MeCP2 may be involved in the establishment and/or maintenance of RME of autosomal genes. To improve our knowledge on this largely unknown phenomenon, and to study the role of MeCP2 in RMAE, we compared RMA gene expression profiles in clonal cell cultures expressing wild-type MeCP2 versus mutant MeCP2 from a RTT patient carrying a pathogenic non-sense variant. Our data clearly demonstrated that MeCP2 deficiency does not affect significantly allelic gene expression of X-linked genes, imprinted genes as well as the RMAE profile in the majority of genes. However, the functional deficiency in MeCP2 appeared to disrupt the mono-allelic or the bi-allelic expression of at least 49 genes allowing us to define a specific signature of MECP2 mutated clones.
Subject(s)
Methyl-CpG-Binding Protein 2/genetics , Rett Syndrome/genetics , Alleles , Allelic Imbalance/genetics , Gene Expression Regulation/genetics , Genes, X-Linked/genetics , Humans , Mutation/genetics , Phenotype , Rett Syndrome/pathologyABSTRACT
MN1 encodes a transcriptional co-regulator without homology to other proteins, previously implicated in acute myeloid leukaemia and development of the palate. Large deletions encompassing MN1 have been reported in individuals with variable neurodevelopmental anomalies and non-specific facial features. We identified a cluster of de novo truncating mutations in MN1 in a cohort of 23 individuals with strikingly similar dysmorphic facial features, especially midface hypoplasia, and intellectual disability with severe expressive language delay. Imaging revealed an atypical form of rhombencephalosynapsis, a distinctive brain malformation characterized by partial or complete loss of the cerebellar vermis with fusion of the cerebellar hemispheres, in 8/10 individuals. Rhombencephalosynapsis has no previously known definitive genetic or environmental causes. Other frequent features included perisylvian polymicrogyria, abnormal posterior clinoid processes and persistent trigeminal artery. MN1 is encoded by only two exons. All mutations, including the recurrent variant p.Arg1295* observed in 8/21 probands, fall in the terminal exon or the extreme 3' region of exon 1, and are therefore predicted to result in escape from nonsense-mediated mRNA decay. This was confirmed in fibroblasts from three individuals. We propose that the condition described here, MN1 C-terminal truncation (MCTT) syndrome, is not due to MN1 haploinsufficiency but rather is the result of dominantly acting C-terminally truncated MN1 protein. Our data show that MN1 plays a critical role in human craniofacial and brain development, and opens the door to understanding the biological mechanisms underlying rhombencephalosynapsis.
Subject(s)
Abnormalities, Multiple/genetics , Craniofacial Abnormalities/genetics , Intellectual Disability/genetics , Language Development Disorders/genetics , Nervous System Malformations/genetics , Trans-Activators/genetics , Tumor Suppressor Proteins/genetics , Abnormalities, Multiple/diagnostic imaging , Adolescent , Basilar Artery/abnormalities , Basilar Artery/diagnostic imaging , Carotid Arteries/abnormalities , Carotid Arteries/diagnostic imaging , Cerebellar Vermis/abnormalities , Cerebellar Vermis/diagnostic imaging , Cerebellum/abnormalities , Cerebellum/diagnostic imaging , Child , Child, Preschool , Cohort Studies , Comparative Genomic Hybridization , Craniofacial Abnormalities/diagnostic imaging , Female , Fibroblasts/metabolism , Humans , Imaging, Three-Dimensional , Infant , Magnetic Resonance Imaging , Male , Middle Aged , Mutation , Nervous System Malformations/diagnostic imaging , Nonsense Mediated mRNA Decay , Polymicrogyria/diagnostic imaging , Polymicrogyria/genetics , RNA-Seq , Real-Time Polymerase Chain Reaction , Syndrome , Tomography, X-Ray Computed , Exome Sequencing , Whole Genome SequencingABSTRACT
PURPOSE: Anorexia nervosa (AN) is a complex neuropsychiatric disorder presenting with dangerously low body weight, and a deep and persistent fear of gaining weight. Up to now, four genome-wide association studies of AN have been conducted to date and identified only few significant loci. However, both previous studies focused on common variation and on rare exonic variants. Currently, de novo variants are one of the most significant risk factors for neurodevelopmental disorders and psychiatric disorders. METHODS: We analyzed by whole exome sequencing a cohort of nine female AN individuals and their parents (mother and father), and focused our analysis on de novo variants. RESULTS: Here, we found seven de novo missense variants in potential genes in nine studied AN patients. Four of these genes (CSMD1, CREB3, PTPRD and GAB1) belong to a same signaling pathway involving neuron differentiation and dopamine pathway. CONCLUSIONS: This study provides a list of interesting genes such as CSDM1 and CREB3 that are candidates to be involved in the etiology of anorexia nervosa. LEVEL OF EVIDENCE: basic research.
Subject(s)
Anorexia Nervosa , Genome-Wide Association Study , Anorexia Nervosa/genetics , Dopamine , Female , Humans , Reward , Exome SequencingABSTRACT
Anorexia nervosa (AN) is a severe debilitating eating disorder. To date, only very few genes that predispose to AN have been identified. An alternative to association studies is to characterize ultra-rare variants in familial forms of AN. Here, we have implemented this approach to identify pathways that contribute to the development of AN through the analysis of a family with three members suffering from AN by exome analysis. We identified three ultra-rare deleterious variants in three genes (DRD4, CCKAR, NMS), already connected to the reward pathway, that co-segregate with AN, suggesting that this pathway might be playing a predisposing role in AN at least in familial forms.
Subject(s)
Anorexia Nervosa/genetics , Adolescent , Adult , Female , Humans , Pedigree , Exome SequencingABSTRACT
BACKGROUND: Anorexia nervosa is a complex neuropsychiatric disorder presenting with life-threatening low body weight, and a persistent fear of gaining weight. To date, no whole exome sequencing was performed in male individuals with anorexia nervosa. AIM AND METHODS: Here, we performed an exome analysis in two independent families with male individuals with anorexia nervosa and found variants in the Neuronatin (NNAT) gene in both probands. To confirm our data, we carried out the screening of the NNAT gene in a cohort of 8 male and 144 female individuals with anorexia nervosa. RESULTS: Exome sequencing revealed a nonsense variant p.Trp33* in NNAT in one patient and a rare variant in the 5'UTR region of NNAT in the other patient. Screening of the NNAT gene in a cohort of 8 male and 144 female individuals with anorexia nervosa allowed to identify 11 other NNAT variants showing that 40.00% and 6.25% of male and female anorexia nervosa individuals carried a NNAT variant, respectively. Moreover, two novel missense variants were identified in female anorexia nervosa patients. CONCLUSION: Our data suggest that NNAT variants and NNAT expression changes may be associated with susceptibility to eating disorders such as anorexia nervosa.
Subject(s)
Anorexia Nervosa/genetics , Genetic Association Studies , Genetic Predisposition to Disease , Membrane Proteins/genetics , Mutation/genetics , Nerve Tissue Proteins/genetics , Exome/genetics , Female , Humans , Inheritance Patterns/genetics , Male , Mutation, Missense/genetics , Pedigree , Exome SequencingABSTRACT
Histone lysine methylation influences processes such as gene expression and DNA repair. Thirty Jumonji C (JmjC) domain-containing proteins have been identified and phylogenetically clustered into seven subfamilies. Most JmjC domain-containing proteins have been shown to possess histone demethylase activity toward specific histone methylation marks. One of these subfamilies, the KDM5 family, is characterized by five conserved domains and includes four members. Interestingly, de novo loss-of-function and missense variants in KDM5B were identified in patients with intellectual disability (ID) and autism spectrum disorder (ASD) but also in unaffected individuals. Here, we report two novel de novo splice variants in the KDM5B gene in three patients with ID and ASD. The c.808â¯+â¯1Gâ¯>â¯A variant was identified in a boy with mild ID and autism traits and is associated with a significant reduced KDM5B mRNA expression without alteration of its H3K4me3 pattern. In contrast, the c.576â¯+â¯2Tâ¯>â¯C variant was found in twins with global delay in developmental milestones, poor language and ASD. This variant causes the production of an abnormal transcript which may produce an altered protein with the loss of the ARID1B domain with an increase in global gene H3K4me3. Our data reinforces the recent observation that the KDM5B haploinsufficiency is not a mechanism involved in intellectual disability and that KDM5B disorder associated with LOF variants is a recessive disorder. However, some variants may also cause gain of function, and need to be interpreted with caution, and functional studies should be performed to identify the molecular consequences of these different rare variants.
Subject(s)
Alternative Splicing , Autism Spectrum Disorder/genetics , Developmental Disabilities/genetics , Intellectual Disability/genetics , Jumonji Domain-Containing Histone Demethylases/genetics , Nuclear Proteins/genetics , Repressor Proteins/genetics , Adolescent , Autism Spectrum Disorder/metabolism , Cell Line , Child , Developmental Disabilities/metabolism , Down-Regulation , Female , Genetic Predisposition to Disease , Haploinsufficiency , Humans , Intellectual Disability/metabolism , Jumonji Domain-Containing Histone Demethylases/metabolism , Male , Nuclear Proteins/metabolism , Pedigree , Repressor Proteins/metabolismABSTRACT
A growing number of histone modifiers are involved in human neurodevelopmental disorders, suggesting that proper regulation of chromatin state is essential for the development of the central nervous system. Among them, heterozygous de novo variants in KMT2A, a gene coding for histone methyltransferase, have been associated with Wiedemann-Steiner syndrome (WSS), a rare developmental disorder mainly characterized by intellectual disability (ID) and hypertrichosis. As KMT2A is known to regulate the expression of multiple target genes through methylation of lysine 4 of histone 3 (H3K4me), we sought to investigate the transcriptomic consequences of KMT2A variants involved in WSS. Using fibroblasts from four WSS patients harboring loss-of-function KMT2A variants, we performed RNA sequencing and identified a number of genes for which transcription was altered in KMT2A-mutated cells compared to the control ones. Strikingly, analysis of the pathways and biological functions significantly deregulated between patients with WSS and healthy individuals revealed a number of processes predicted to be altered that are relevant for hypertrichosis and intellectual disability, the cardinal signs of this disease.
Subject(s)
Histone-Lysine N-Methyltransferase/genetics , Histones/metabolism , Hypertrichosis/genetics , Hypertrichosis/metabolism , Intellectual Disability/genetics , Intellectual Disability/metabolism , Myeloid-Lymphoid Leukemia Protein/genetics , Rare Diseases/genetics , Rare Diseases/metabolism , Adolescent , Adult , Child , Female , Fibroblasts , Gene Expression Regulation , Humans , Loss of Function Mutation , Lysine/metabolism , Male , Methylation , Middle Aged , Nitric Oxide Synthase Type III/metabolism , Sequence Analysis, RNA , Signal Transduction , Syndrome , Transcription, Genetic , Young AdultABSTRACT
Genetic findings reported by our group and others showed that de novo missense variants in the KIF2A gene underlie malformations of brain development called pachygyria and microcephaly. Though KIF2A is known as member of the Kinesin-13 family involved in the regulation of microtubule end dynamics through its ATP dependent MT-depolymerase activity, how KIF2A variants lead to brain malformations is still largely unknown. Using cellular and in utero electroporation approaches, we show here that KIF2A disease-causing variants disrupts projection neuron positioning and interneuron migration, as well as progenitors proliferation. Interestingly, further dissection of this latter process revealed that ciliogenesis regulation is also altered during progenitors cell cycle. Altogether, our data suggest that deregulation of the coupling between ciliogenesis and cell cycle might contribute to the pathogenesis of KIF2A-related brain malformations. They also raise the issue whether ciliogenesis defects are a hallmark of other brain malformations, such as those related to tubulins and MT-motor proteins variants.
Subject(s)
Cilia/genetics , Kinesins/metabolism , Malformations of Cortical Development/genetics , Repressor Proteins/metabolism , Animals , Brain/metabolism , Cell Cycle/genetics , Cilia/physiology , HeLa Cells , Humans , Kinesins/genetics , Malformations of Cortical Development/metabolism , Mice , Microcephaly/metabolism , Microtubules/metabolism , Neurogenesis , Repressor Proteins/genetics , Spindle Apparatus/metabolism , Tubulin/metabolismABSTRACT
Variants in KMT2A, encoding the histone methyltransferase KMT2A, are a growing cause of intellectual disability (ID). Up to now, the majority of KMT2A variants are non-sense and frameshift variants causing a typical form of Wiedemann-Steiner syndrome. We studied KMT2A gene in a cohort of 200 patients with unexplained syndromic and non-syndromic ID and identified four novel variants, one splice and three missense variants, possibly deleterious. We used primary cells from the patients and molecular approaches to determine the deleterious effects of those variants on KMT2A expression and function. For the putative splice variant c.11322-1G>A, we showed that it led to only one nucleotide deletion and loss of the C-terminal part of the protein. For two studied KMT2A missense variants, c.3460C>T (p.(Arg1154Trp)) and c.8558T>G (p.(Met2853Arg)), located at the cysteine-rich CXXC domain and the transactivation domain of the protein, respectively, we found altered KMT2A target genes expression in patient's fibroblasts compared to controls. Furthermore, we found a disturbed subcellular distribution of KMT2A for the c.3460C>T mutant. Taken together, our results demonstrated the deleterious impact of the splice variant and of the missense variants located at two different functional domains and suggested reduction of KMT2A function as the disease-causing mechanism.
Subject(s)
Abnormalities, Multiple/genetics , Histone-Lysine N-Methyltransferase/genetics , Hypertrichosis/genetics , Intellectual Disability/genetics , Myeloid-Lymphoid Leukemia Protein/genetics , Abnormalities, Multiple/pathology , Animals , COS Cells , Cells, Cultured , Child, Preschool , Chlorocebus aethiops , Female , Histone-Lysine N-Methyltransferase/metabolism , Humans , Hypertrichosis/pathology , Infant , Intellectual Disability/pathology , Loss of Function Mutation , Male , Mutation, Missense , Myeloid-Lymphoid Leukemia Protein/metabolism , RNA Splicing , SyndromeABSTRACT
Neurodevelopmental disorders with periventricular nodular heterotopia (PNH) are etiologically heterogeneous, and their genetic causes remain in many cases unknown. Here we show that missense mutations in NEDD4L mapping to the HECT domain of the encoded E3 ubiquitin ligase lead to PNH associated with toe syndactyly, cleft palate and neurodevelopmental delay. Cellular and expression data showed sensitivity of PNH-associated mutants to proteasome degradation. Moreover, an in utero electroporation approach showed that PNH-related mutants and excess wild-type NEDD4L affect neurogenesis, neuronal positioning and terminal translocation. Further investigations, including rapamycin-based experiments, found differential deregulation of pathways involved. Excess wild-type NEDD4L leads to disruption of Dab1 and mTORC1 pathways, while PNH-related mutations are associated with deregulation of mTORC1 and AKT activities. Altogether, these data provide insights into the critical role of NEDD4L in the regulation of mTOR pathways and their contributions in cortical development.