Rare deleterious mutations of HNRNP genes result in shared neurodevelopmental disorders

Familias de genes; Trastornos del desarrollo neurológico; HnRNPsFamílies genètiques; Trastorns del desenvolupament neurològic; HnRNPsGene families; Neurodevelopmental disorders; HnRNPsBackground With the increasing number of genomic sequencing studies, hundreds of genes have been implicated in neurodevelopmental disorders (NDDs). The rate of gene discovery far outpaces our understanding of genotype–phenotype correlations, with clinical characterization remaining a bottleneck for understanding NDDs. Most disease-associated Mendelian genes are members of gene families, and we hypothesize that those with related molecular function share clinical presentations. Methods We tested our hypothesis by considering gene families that have multiple members with an enrichment of de novo variants among NDDs, as determined by previous meta-analyses. One of these gene families is the heterogeneous nuclear ribonucleoproteins (hnRNPs), which has 33 members, five of which have been recently identified as NDD genes (HNRNPK, HNRNPU, HNRNPH1, HNRNPH2, and HNRNPR) and two of which have significant enrichment in our previous meta-analysis of probands with NDDs (HNRNPU and SYNCRIP). Utilizing protein homology, mutation analyses, gene expression analyses, and phenotypic characterization, we provide evidence for variation in 12 HNRNP genes as candidates for NDDs. Seven are potentially novel while the remaining genes in the family likely do not significantly contribute to NDD risk. Results We report 119 new NDD cases (64 de novo variants) through sequencing and international collaborations and combined with published clinical case reports. We consider 235 cases with gene-disruptive single-nucleotide variants or indels and 15 cases with small copy number variants. Three hnRNP-encoding genes reach nominal or exome-wide significance for de novo variant enrichment, while nine are candidates for pathogenic mutations. Comparison of HNRNP gene expression shows a pattern consistent with a role in cerebral cortical development with enriched expression among radial glial progenitors. Clinical assessment of probands (n = 188–221) expands the phenotypes associated with HNRNP rare variants, and phenotypes associated with variation in the HNRNP genes distinguishes them as a subgroup of NDDs. Conclusions Overall, our novel approach of exploiting gene families in NDDs identifies new HNRNP-related disorders, expands the phenotypes of known HNRNP-related disorders, strongly implicates disruption of the hnRNPs as a whole in NDDs, and supports that NDD subtypes likely have shared molecular pathogenesis. To date, this is the first study to identify novel genetic disorders based on the presence of disorders in related genes. We also perform the first phenotypic analyses focusing on related genes. Finally, we show that radial glial expression of these genes is likely critical during neurodevelopment. This is important for diagnostics, as well as developing strategies to best study these genes for the development of therapeutics.This work was supported, in part, by the U.S. National Institutes of Health (R01MH101221) to E.E.E. Research reported in this publication was supported, in part, by the National Institute of Neurological Disorders and Stroke (NINDS) under award number K08NS092898, Jordan’s Guardian Angels, and the Brotman Baty Institute (to G.M.M.). M.I., A.C., and A.S. were supported by the G.E.N.E. (Genomic analysis Evaluation Network) Research Project founded by Progetti di Innovazione in Ambito Sanitario e Socio Sanitario (Bando EX decreto n.2713 28.02.2018) Regione Lombardia. D. L was supported by the German Research Foundation (DFG; LE 4223/1). B.B.A.d.V. and L.E.L.M.V. were supported by grants from the Dutch Organization for Health Research and Development (ZON-MW grants 917–86–319 and 912–12–109). M.E., O.G., and C.R. received funding from the Italian Ministry of Health (Project RC n. 2757328). I.T. is supported by generous donors to the Children’s Mercy Research Institute and the Genomic Answers for Kids program. K.X. is supported by the National Natural Science Foundation of China (NSFC: 8173000779) and the Science and Technology Major Project of Hunan Provincial Science and Technology Department (2018SK1030). M.A.G. was supported by the U.S. National Institutes of Health (T32HG000035). E.E.E. is an investigator of the Howard Hughes Medical Institute

Clinical and Radiological Markers of Extra-Motor Deficits in Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is now universally recognized as a complex multisystem disorder with considerable extra-motor involvement. The neuropsychological manifestations of frontotemporal, parietal, and basal ganglia involvement in ALS have important implications for compliance with assistive devices, survival, participation in clinical trials, caregiver burden, and the management of individual care needs. Recent advances in neuroimaging have been instrumental in characterizing the biological substrate of heterogeneous cognitive and behavioral deficits in ALS. In this review we discuss the clinical and radiological aspects of cognitive and behavioral impairment in ALS focusing on the recognition, assessment, and monitoring of these symptoms

A P300 Based Cognitive Assessment Battery for Severely Motor-impaired and Overtly Non-responsive Patients

Diagnosing disorders of consciousness (DOC) is notoriously difficult, with estimates of misdiagnosis rates as high as 40%. Moreover, recent studies have demonstrated that patients who do not show signs of volitional motor responses can exhibit preserved command following detected by functional magnetic resonance imaging (fMRI) and electroencephalography (EEG). Although these patients clearly retain some cognitive abilities, lack of consistent motor responses makes administration of standard neuropsychological tests impossible. Consequently, the extent of their cognitive function is unknown. In the current study, we developed and validated a P300b event related potential (ERP) neuropsychological battery in healthy participants to assess components of executive function without requiring motor output. First, participants were instructed to attend to a target auditory stimulus. P300b responses to attended relative to unattended stimuli were used as a neural proxy for detecting command following. To assess working memory capacity we adapted a digit span test to use a similar P300b response mechanism. Finally, reasoning was assessed by adapting a verbal reasoning task in the same manner. At the group level, and in a large majority of participants at the single-participant level, accurate performance could be detected using the P300b ERP, validating the potential utility of the battery. Additionally, the normalized magnitude of the P300b predicted individual differences in performance, but only when a suitable level of variability between participants was present. A post hoc Monte Carlo analysis was conducted to examine the necessary time required to conduct the battery as well as the interaction between time and performance in determining statistically significant performance. At 100% accuracy, a mean time of five minutes was required to achieve a significant result, with time increasing as a function of decreasing performance. These results demonstrate that covert control of attention, as measured by the P300b ERP, can be used to assess command following, working memory and reasoning abilities with a high degree of reliabilit

Verb Use in Parkinson\u27s Disease

Verb-specific impairments in their use and comprehension are well documented in persons with Parkinson’s disease (PD). The grammatical and the motor theories have been proposed as possible explanations for verb impairments. The purpose of this study is to describe the use of low-motion and high-motion content verbs in PD in everyday conversation and to determine which theory best supports these findings. In this cross-sectional prospective study, conversation samples were collected and analyzed from participants with PD and their spouses in a mealtime context. Results indicated that total verb use on a proportional basis was not significantly different between persons with PD vs. control participants. Participants with PD produced significantly fewer high-motion verbs compared to low-motion content verbs. However, control participants also produced significantly fewer high-motion verbs compared to low-motion content verbs. The findings do not support the motor theory or the grammatical theory of verb processing in participants with PD

Neuronal network disintegration: common pathways linking neurodegenerative diseases

Neurodegeneration refers to a heterogeneous group of brain disorders that progressively evolve. It has been increasingly appreciated that many neurodegenerative conditions overlap at multiple levels and therefore traditional clinicopathological correlation approaches to better classify a disease have met with limited success. Neuronal network disintegration is fundamental to neurodegeneration, and concepts based around such a concept may better explain the overlap between their clinical and pathological phenotypes. In this Review, promoters of overlap in neurodegeneration incorporating behavioural, cognitive, metabolic, motor, and extrapyramidal presentations will be critically appraised. In addition, evidence that may support the existence of large-scale networks that might be contributing to phenotypic differentiation will be considered across a neurodegenerative spectrum. Disintegration of neuronal networks through different pathological processes, such as prion-like spread, may provide a better paradigm of disease and thereby facilitate the identification of novel therapies for neurodegeneration

Graph theory applied to neuroimaging data reveals key functional connectivity alterations in brain of behavioral variant Frontotemporal Dementia subjects

Brain functional architecture and anatomical structure have been intensively studied to generate efficient models of its complex mechanisms. Functional alterations and cognitive impairments are the most investigated aspects in the recent clinical research as distinctive traits of neurodegeneration. Although specific behaviours are clearly associated to neurodegeneration, information flow breakdown within the brain functional network, responsible to deeply affect cognitive skills, remains not completely understood. Behavioural variant Frontotemporal Dementia (bvFTD) is the most common type of Frontotemporal degeneration, marked by behavioural disturbances, social instabilities and impairment of executive functions. Mathematical modelling offers effective tools to inspect deviations from physiological cognitive functions and connectivity alterations. As a popular recent methodology, graph theoretical approaches applied to imaging data expanded our knowledge of neurodegenerative disorders, although the need for unbiased metrics is still an open issue. In this thesis, we propose an integrated analysis of functional features among brain areas in bvFTD patients, to assess global connectivity and topological network alterations respect to the healthy condition, using a minimum spanning tree (MST) based-model to resting state functional MRI (rs-fMRI) data. Contrary to several graph theoretical approaches, dependent to arbitrary criteria (e.g., correlation thresholds, network density or a priori distribution), MST represents an unambiguous modelling solution, ensuring full reproducibility and robustness in different conditions. Our MSTs were obtained from wavelet correlation matrices derived from mean time series intensities, extracted from 116 regions of interest (ROIs) of 41 bvFTD patients and 39 healthy controls (HC), which underwent rs-fMRI. The resulting graphs were tested for global connectivity and topological differences between the two groups, by applying a Wilcoxon rank sum test with a significance level at 0.05 (nonparametric median difference estimates with 95% confidence interval). The same test was applied for methodological comparison between MST and other common graph theory methods. After methodological comparisons, our MST model achieved the best bvFTD/HC separation performances, without a priori assumptions. Direct MST comparison between bvFTD and healty controls revealed key brain functional architecture differences. Diseased subjects showed a linear-shape network configuration tendency, with high distance between nodes, low centrality parameter values, and a low exchange information capacity (i.e., low network integration) in MST parameters. Moreover, edge-level and node-level features (i.e., superhighways, and node degree and betweenness centrality) indicated a more complex scenario, showing some of the key bvFTD dysfunctions observed in large scale resting-state functional networks (default-mode (DMN), salience (SN), and executive (EN) networks), suggesting an underlying involvement of the limbic system in the observed functional deterioration. Functional isolation has been observed as a generalized process affecting the entire bvFTD network, showing brain macro-regions isolation, with homogeneous functional distribution of brain areas, longer distances between hubs, and longer within-lobe superhighways. Conversely, the HC network showed marked functional integration, where superhighways serve as shortcuts to connect areas from different brain macro-regions. The combination of this theoretical model with rs-fMRI data constitutes an effective method to generate a clear picture of the functional divergence between bvFTD and HCs, providing possible insights on the effects of frontotemporal neurodegeneration and compensatory mechanisms underlying characteristic bvFTD cognitive, social, and executive impairments

Analysis of candidate genes for behavioral differences in mice

Organisms have evolved different behavioral strategies for better survival and reproduction. However, the genetic basis for such traits remains still as a longstanding fascinating question in evolutionary biology. Mate choice strategy is one of the behavioral traits which can play an important role in the life history of organisms. A previous study had shown that mate choice preference between two populations of the Western house mouse (M. m. domesticus) is influenced by the genetic background of the fathers. Transcriptome analysis in a follow up study revealed an imprinted cluster on Chromosome 7, known as Prader-Willi Syndrome (PWS) region, and also Peg13 on Chromosome 15 as loci that are highly differentiated between mouse populations and therefore have been suggested as potential regions which may regulate this paternal preference in the house mouse. The present thesis was aimed to investigate the functional role of these two imprinted regions in Western house mice behavior. In the first chapter, I investigated the role of the PWS region on Chromosome 7 through a variety of techniques, including copy number variation analysis, behavioral correlations and transcriptomics. I found that two paternally imprinted tandemly repeated regulatory RNA coding genes (SNORD115 and SNORD116) are of special interest. Their copy number evolves very fast and correlates highly with personality traits between individuals. Further I found that the copy number variation influences the expression of more than 130 genes including genes involved in serotonin regulation, vocalization and bone development. The findings suggest a molecular mechanism for the generation and variability of personality traits in mammals. The second chapter focuses on the analysis of paternally imprinted Peg13 gene, which has so far not been functionally studied. This gene has been known as non-coding gene, but data from ribosomal profiling analysis revealed that this gene could indeed produce a small peptide and it is not a simple non-coding gene. By using knock out mice, I showed even a partial deletion at 3’ part of Peg13 could significantly change expression of hundreds genes in the brain and could thus influence various mouse behaviors. The phenotypic analysis showed a significant effect on mouse sexual and parental behavior. Since RNAseq analysis from mouse brain development timeline showed that this gene has highest expression in day 12.5, I propose that Peg13 may play important role in preoptic area development with possible direct role in sexual and mate choice behavior. 10 Overall the work presented in this thesis describes the first major molecular mechanism underlying mouse personality traits. It presents also the first functional study on the Peg13 gene in mice, which highlights its possible role in mouse brain development and sexual behavior.Contents Summary ................................................................................................................................................. 9 Zusammenfassung ................................................................................................................................. 11 General introduction ............................................................................................................................. 14 Behavior and evolution ..................................................................................................................... 14 Mate choice ....................................................................................................................................... 14 Assortative mating ............................................................................................................................ 15 House mouse and paternal mate choice preference .......................................................................... 16 1-Competitive Ability .................................................................................................................... 17 2-Personality ................................................................................................................................. 18 3-Ultrasonic Vocalization ................................................................................................................. 18 Genomic imprinting .......................................................................................................................... 19 Genomic imprinting and brain development .................................................................................... 19 Imprinted gene and mate choice preference in house mouse ............................................................ 20 Aim of the project ............................................................................................................................. 22 Chapter1 ................................................................................................................................................ 24 Involvement of the Prader-Willi Syndrome (PWS) region genes in mouse behavior .......................... 24 Introduction ....................................................................................................................................... 25 Analysis of SNORD Copy Number Variation .................................................................................. 28 SNORD115 and 116 target genes ..................................................................................................... 30 Link between SNORD copy number and personality ....................................................................... 34 Correlation between SNORD115 and 116 copy numbers ................................................................ 42 Inheritance of SNORD copy numbers .............................................................................................. 45 SNORD copy number differences across tissues .............................................................................. 50 Variation of personality in inbred mice ............................................................................................ 51 SNORD copy number influence on the brain transcriptome ............................................................ 54 SNORD116 copy number and craniofacial features ......................................................................... 60 Differential gene expression through SNORD CNV ........................................................................ 64 Discussion ......................................................................................................................................... 66 SNROD115/ 116 CNV and personality ............................................................................................ 66 Correlation of personality and cognitive ability ............................................................................... 67 Correlation of personality and metabolism ....................................................................................... 68 Correlation of personality traits and vocalization ............................................................................. 68 Personality and craniofacial features ................................................................................................ 69 SNOD115/ 116 and the Prader-Willi syndrome ............................................................................... 69 6 Inheritance of SNROD115 and 116 .................................................................................................. 70 Mice sample ...................................................................................................................................... 71 Mouse keeping .................................................................................................................................. 71 Mouse dissection ............................................................................................................................... 71 DNA extraction ................................................................................................................................. 71 mRNA extraction and cDNA synthesis ............................................................................................ 72 RNAseq analysis ............................................................................................................................... 73 Small RNA extraction, cDNA synthesis, library preparation and sequencing ................................. 73 Read mapping ................................................................................................................................... 74 snoRNA analysis ............................................................................................................................... 75 Droplet digital PCR ........................................................................................................................... 75 1- Finding a suitable reference gene ............................................................................................ 76 2-Primer designing ....................................................................................................................... 76 3-DNA digestion and ddPCR ........................................................................................................ 77 4-Copy Number Calculation ......................................................................................................... 78 Mouse Personality ............................................................................................................................. 81 Behavioral Tests................................................................................................................................ 81 Elevated Plus Maze ........................................................................................................................... 81 Open Field ......................................................................................................................................... 81 Dark/Light Box ................................................................................................................................. 81 Statistical Analysis ............................................................................................................................ 83 Shape phenotyping ............................................................................................................................ 85 Chapter 2 ............................................................................................................................................... 90 Functional analysis of the paternally expressed gene Peg13 ................................................................ 90 Introduction ....................................................................................................................................... 91 Results ............................................................................................................................................... 93 Peg13 and Trappc9 Expression ........................................................................................................ 93 Peg13 expression in the brain ........................................................................................................... 96 Peg13 expression during developmental stages ................................................................................ 97 A possible PEG13 protein ................................................................................................................. 98 Generation of Knock-out mice .......................................................................................................... 99 Peg13 3’-knockout mouse .............................................................................................................. 101 Behavioral tests on Peg13 3’-region knockout mice ...................................................................... 102 RNAseq analysis on Peg13 3’- region knockout mice ................................................................... 108 GO analysis ..................................................................................................................................... 109 7 Discussion ....................................................................................................................................... 109 Expression of Peg13, Kcnk9 and Trappc9 are co-regulated ........................................................... 109 Peg13 is not a simple non-coding RNA .......................................................................................... 110 Behavior of Peg13 3’-region knockout mice .................................................................................. 110 Anxiety behavior of Peg13 3’-KO mice ......................................................................................... 111 Conclusion ...................................................................................................................................... 112 Material and Methods ..................................................................................................................... 113 RNAseq analysis across different tissues ........................................................................................ 113 Ribosomal profiling data analysis ................................................................................................... 113 Generation of knock-out mice by genOway ................................................................................... 113 Novel Object Test ........................................................................................................................... 114 Other procedures ............................................................................................................................. 114 General Discussion ............................................................................................................................. 116 The general background underlying this PhD thesis....................................................................... 116 The PWS region may be associated to the paternal mate choice preference in Western house mouse by regulating mouse personality ..................................................................................................... 117 Evolution of personality .................................................................................................................. 117 1-Selective Neutrality .................................................................................................................. 117 2-Mutation-selection balance ..................................................................................................... 118 3-Balancing Selection ................................................................................................................. 122 Personality and imprinting .............................................................................................................. 123 1-Different recombination rate between male and female .......................................................... 123 2-Co-adapted gene expression .................................................................................................... 123 Peg13 could be involved in sexual behavior in mouse ................................................................... 124 Conclusion .......................................................................................................................................... 126 Reference ............................................................................................................................................ 128 Acknowledgements ............................................................................................................................. 151 Contributions to the thesis .................................................................................................................. 153 Declaration .......................................................................................................................................... 154 Curriculum Vitae ................................................................................................................................ 15