Background: The cardiomyopathies, classically categorized as hypertrophic (HCM), dilated (DCM), and arrhythmogenic right ventricular (ARVC), each have a signature genetic theme. HCM and ARVC are largely understood as genetic diseases of sarcomere or desmosome proteins, respectively. In contrast, >250 genes spanning more than 10 gene ontologies have been implicated in DCM, representing a complex and diverse genetic architecture. To clarify this, a systematic curation of evidence to establish the relationship of genes with DCM was conducted. Methods: An international Panel with clinical and scientific expertise in DCM genetics evaluated evidence supporting monogenic relationships of genes with idiopathic DCM. The Panel utilized the ClinGen semi-quantitative gene-disease clinical validity classification framework with modifications for DCM genetics to classify genes into categories based on the strength of currently available evidence. Representation of DCM genes on clinically available genetic testing panels was evaluated. Results: Fifty-one genes with human genetic evidence were curated. Twelve genes (23%) from eight gene ontologies were classified as having definitive ( BAG3, DES, FLNC, LMNA, MYH7, PLN, RBM20, SCN5A, TNNC1, TNNT2, TTN ) or strong ( DSP ) evidence. Seven genes (14%) ( ACTC1, ACTN2, JPH2, NEXN, TNNI3, TPM1, VCL ) including two additional ontologies were classified as moderate evidence; these genes are likely to emerge as strong or definitive with additional evidence. Of these 19 genes, six were similarly classified for HCM and three for ARVC. Of the remaining 32 genes (63%), 25 (49%) had limited evidence, 4 (8%) were disputed, 2 (4%) had no disease relationship, and 1 (2%) was supported by animal model data only. Of 16 evaluated clinical genetic testing panels, most definitive genes were included, but panels also included numerous genes with minimal human evidence. Conclusions: In the curation of 51 genes, 19 had high evidence (12 definitive/strong; seven moderate). Notably, these 19 genes only explain a minority of cases, leaving the remainder of DCM genetic architecture incompletely addressed. Clinical genetic testing panels include most high evidence genes, however genes lacking robust evidence are also commonly included. We recommend that high evidence DCM genes be used for clinical practice and to exercise caution when interpreting variants in variable evidence DCM genes.

Precise control of messenger RNA (mRNA) processing and abundance are increasingly being recognized as critical for proper spatiotemporal gene expression, particularly in neurons. These regulatory events are governed by a large number of transacting factors found in neurons, most notably RNA-binding proteins (RBPs) and micro-RNAs (miRs), which bind to specific cisacting elements or structures within mRNAs. Through this binding mechanism, trans-acting factors, particularly RBPs, control all aspects of mRNA metabolism, ranging from altering the transcription rate to mediating mRNA degradation. In this context the best-characterized neuronal RBP, the Hu/ELAVl family member HuD, is emerging as a key component in multiple regulatory processes-including pre-mRNA processing, mRNA stability, and translation-governing the fate of a substantial amount of neuronal mRNAs. Through its ability to regulate mRNA metabolism of diverse groups of functionally similar genes, HuD plays important roles in neuronal development and function. Furthermore, compelling evidence indicates supplementary roles for HuD in neuronal plasticity, in particular, recovery from axonal injury, learning and memory, and multiple neurological diseases. The purpose of this review is to provide a detailed overview of the current knowledge surrounding the expression and roles of HuD in the nervous system. Additionally, we outline the present understanding of the molecular mechanisms presiding over the localization, abundance, and function of HuD in neurons.

Background: The cardiomyopathies are classically categorized as hypertrophic (HCM), dilated (DCM), and arrhythmogenic right ventricular (ARVC), and each have a signature genetic theme. HCM and ARVC are largely understood as genetic diseases of sarcomere or desmosome proteins, respectively. In contrast, >250 genes spanning more than 10 gene ontologies have been implicated in DCM, representing a complex and diverse genetic architecture. To clarify this, a systematic curation of evidence to establish the relationship of genes with DCM was conducted. Methods: An international Panel with clinical and scientific expertise in DCM genetics was assembled to evaluate evidence supporting monogenic relationships of genes with idiopathic DCM. The Panel utilized the ClinGen semi-quantitative gene-disease clinical validity classification framework. Results: Fifty-one genes with human genetic evidence were curated. Twelve genes (23%) from eight gene ontologies were classified as having definitive (BAG3, DES, FLNC, LMNA, MYH7, PLN, RBM20, SCN5A, TNNC1, TNNT2, TTN) or strong (DSP) evidence. Seven genes (14%) (ACTC1, ACTN2, JPH2, NEXN, TNNI3, TPM1, VCL) including two additional ontologies were classified as moderate evidence; these genes are likely to emerge as strong or definitive with additional evidence. Of the 19 genes classified as definitive, strong or moderate, six were similarly classified for HCM and three for ARVC. Of the remaining 32 genes (63%), 25 (49%) had limited evidence, 4 (8%) were disputed, 2 (4%) had no disease relationship, and 1 (2%) was supported by animal model data only. Of 16 commercially available genetic testing panels evaluated, most definitive genes were included, but panels also included numerous genes with minimal human evidence. Conclusions: In a systematic curation of published evidence for genes considered relevant for monogenic DCM, 12 were classified as definitive or strong and seven as moderate evidence spanning 10 gene ontologies. Notably, these 19 genes only explain a minority of DCM cases, leaving the remainder of DCM genetic architecture incompletely addressed. While clinical genetic testing panels include most high evidence genes, genes lacking robust evidence are also commonly included. Until the genetic architecture of DCM is more fully defined, care should be taken in the interpretation of variable evidence DCM genes in clinical practice.

The dual-specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A) gene, located on chromosome 21q22.13 within the Down syndrome critical region, has been implicated in syndromic intellectual disability associated with Down syndrome and autism. DYRK1A has a critical role in brain growth and development primarily by regulating cell proliferation, neurogenesis, neuronal plasticity and survival. Several patients have been reported with chromosome 21 aberrations such as partial monosomy, involving multiple genes including DYRK1A. In addition, seven other individuals have been described with chromosomal rearrangements, intragenic deletions or truncating mutations that disrupt specifically DYRK1A. Most of these patients have microcephaly and all have significant intellectual disability. In the present study, we report 10 unrelated individuals with DYRK1A-associated intellectual disability (ID) who display a recurrent pattern of clinical manifestations including primary or acquired microcephaly, ID ranging from mild to severe, speech delay or absence, seizures, autism, motor delay, deep-set eyes, poor feeding and poor weight gain. We identified unique truncating and non-synonymous mutations (three nonsense, four frameshift and two missense) in DYRK1A in nine patients and a large chromosomal deletion that encompassed DYRK1A in one patient. On the basis of increasing identification of mutations in DYRK1A, we suggest that this gene be considered potentially causative in patients presenting with ID, primary or acquired microcephaly, feeding problems and absent or delayed speech with or without seizures.

Upregulation of utrophin A is an attractive therapeutic strategy for treating Duchenne muscular dystrophy (DMD). Over the years, several studies revealed that utrophin A is regulated by multiple transcriptional and post-transcriptional mechanisms, and that pharmacological modulation of these pathways stimulates utrophin A expression in dystrophic muscle. In particular, we recently showed that activation of p38 signaling causes an increase in the levels of utrophin A mRNAs and protein by decreasing the functional availability of the destabilizing RNA-binding protein called K-homology splicing regulatory protein, thereby resulting in increases in the stability of existing mRNAs. Here, we treated 6-week-old mdx mice for 4 weeks with the clinically used anticoagulant drug heparin known to activate p38 mitogen-activated protein kinase, and determined the impact of this pharmacological intervention on the dystrophic phenotype. Our results show that heparin treatment of mdx mice caused a significant ∼1.5- to 3-fold increase in utrophin A expression in diaphragm, extensor digitorum longus and tibialis anterior (TA) muscles. In agreement with these findings, heparin-treated diaphragm and TA muscle fibers showed an accumulation of utrophin A and β-dystroglycan along their sarcolemma and displayed improved morphology and structural integrity. Moreover, combinatorial drug treatment using both heparin and 5-amino-4-imidazolecarboxamide riboside (AICAR), the latter targeting 5' adenosine monophosphate-activated protein kinase and the transcriptional activation of utrophin A, caused an additive effect on utrophin A expression in dystrophic muscle. These findings establish that heparin is a relevant therapeutic agent for treating DMD, and illustrate that combinatorial treatment of heparin with AICAR may serve as an effective strategy to further increase utrophin A expression in dystrophic muscle via activation of distinct signaling pathways.

PURPOSE: DYRK1A syndrome is among the most frequent monogenic forms of intellectual disability (ID). We refined the molecular and clinical description of this disorder and developed tools to improve interpretation of missense variants, which remains a major challenge in human genetics. METHODS: We reported clinical and molecular data for fifty individuals with ID harboring DYRK1A variants and developed i) a specific DYRK1A clinical score, ii) amino acid conservation data generated from one hundred of DYRK1A sequences across different taxa, iii) in vitro overexpression assays to study level, cellular localization, and kinase activity of DYRK1A mutant proteins, and iv) a specific blood DNA methylation signature. RESULTS: This integrative approach was successful to reclassify several variants as pathogenic. However, we questioned the involvement of some others, such as p.Thr588Asn, still reported as likely pathogenic, and showed it does not cause an obvious phenotype in mice. CONCLUSION: Our study demonstrated the need for caution when interpreting variants in DYRK1A, even those occurring de novo. The tools developed will be useful to interpret accurately the variants identified in the future in this gene. ACCEPTED MANUSCRIPT -CLEAN COPYand analyzed as previously described 20 , a total of n=774,590 probes were analyzed for differential methylation. Standard quality control metrics showed good data quality for all samples except Ind#20. Briefly, limma regression with covariates age, sex, and five predicted blood cell types identified a DNAm signature with a Benjamini-Hochberg adjusted p-value<0.05 and 10% methylation difference. Next, we developed a support vector machine (SVM) model with linear kernel trained on including non-redundant CpG sites 20 using the methylation values for the discovery cases vs. controls. The model generated scores ranging between 0 and 1 for tested samples, classifying samples as "positive" (score>0.5) or "negative" (score<0.5). Additional neurotypical controls (n=94) and DYRK1A LoF validation samples (n=6) were scored to test model specificity and sensitivity respectively, and samples with pathogenic KMT2A (n=8) and ARID1B (n=4) variants and DYRK1A missense and distal frameshift (n=11) variants were tested. RESULTS Identification of genetic variants in DYRK1A in individuals with IDWe collected molecular and clinical information from 50 individuals with ID (44 never reported and six previously reported 6,7 ) carrying a variant in DYRK1A identified in clinical and diagnostic laboratories: structural variants deleting or interrupting DYRK1A and recurrent or novel nonsense, frameshift, splice and missense variants (Table 1, Figure S1). When blood or fibroblast samples were available, we characterized the consequences of these variants on DYRK1A mRNA by RNA-sequencing and RT-qPCR (Figure S2, Supplementary Text). For one variant, c.1978del, occurring in the last exon of the gene (Ind #18), the mutant transcripts escape to nonsense mRNA mediated decay (NMD) and result in a truncated protein p.Se...

The RBP (RNA-binding protein) and Hu/ELAV family member HuD regulates mRNA metabolism of genes directly or indirectly involved in neuronal differentiation, learning and memory, and several neurological diseases. Given the important functions of HuD in a variety of processes, we set out to determine the mechanisms that promote HuD mRNA expression in neurons using a mouse model. Through several complementary approaches, we determined that the abundance of HuD mRNA is predominantly under transcriptional control in developing neurons. Bioinformatic and 5ЈRACE (rapid amplification of cDNA ends) analyses of the 5Ј genomic flanking region identified eight conserved HuD leader exons (E1s), two of which are novel. Expression of all E1 variants was determined in mouse embryonic (E14.5) and adult brains. Sequential deletion of the 5Ј regulatory region upstream of the predominantly expressed E1c variant revealed a well conserved 400 bp DNA region that contains five E-boxes and is capable of directing HuD expression specifically in neurons. Using EMSA (electrophoretic mobility shift assay), ChIP (chromatin immunoprecipitation), and 5Ј regulatory region deletion and mutation analysis, we found that two of these E-boxes are targets of Neurogenin 2 (Ngn2) and that this mechanism is important for HuD mRNA induction. Together, our findings reveal that transcriptional regulation of HuD involves the use of alternate leader exons and Ngn2 mediates neuron-specific mRNA expression. To our knowledge, this is the first study to identify molecular events that positively regulate HuD mRNA expression.

Up-regulation of utrophin in muscles represents a promising therapeutic strategy for the treatment of Duchenne Muscular Dystrophy. We previously demonstrated that eEF1A2 associates with the 5'UTR of utrophin A to promote IRES-dependent translation. Here, we examine whether eEF1A2 directly regulates utrophin A expression and identify via an ELISAbased high-throughput screen, FDA-approved drugs that upregulate both eEF1A2 and utrophin A. Our results show that transient overexpression of eEF1A2 in mouse muscles causes an increase in IRES-mediated translation of utrophin A. Through the assessment of our screen, we reveal 7 classes of FDA-approved drugs that increase eEF1A2 and utrophin A protein levels. Treatment of mdx mice with the 2 top leads results in multiple improvements of the dystrophic phenotype. Here, we report that IRES-mediated translation of utrophin A via eEF1A2 is a critical mechanism of regulating utrophin A expression and reveal the potential of repurposed drugs for treating DMD via this pathway.