The neuromuscular junction (NMJ) is one of the best-studied cholinergic synapses. Inherited defects of peripheral neurotransmission result in congenital myasthenic syndromes (CMSs), a clinically and genetically heterogeneous group of rare diseases with fluctuating fatigable muscle weakness as the clinical hallmark. Whole-exome sequencing and Sanger sequencing in six unrelated families identified compound heterozygous and homozygous mutations in SLC5A7 encoding the presynaptic sodium-dependent high-affinity choline transporter 1 (CHT), which is known to be mutated in one dominant form of distal motor neuronopathy (DHMN7A). We identified 11 recessive mutations in SLC5A7 that were associated with a spectrum of severe muscle weakness ranging from a lethal antenatal form of arthrogryposis and severe hypotonia to a neonatal form of CMS with episodic apnea and a favorable prognosis when well managed at the clinical level. As expected given the critical role of CHT for multisystemic cholinergic neurotransmission, autonomic dysfunctions were reported in the antenatal form and cognitive impairment was noticed in half of the persons with the neonatal form. The missense mutations induced a near complete loss of function of CHT activity in cell models. At the human NMJ, a delay in synaptic maturation and an altered maintenance were observed in the antenatal and neonatal forms, respectively. Increased synaptic expression of butyrylcholinesterase was also observed, exposing the dysfunction of cholinergic metabolism when CHT is deficient in vivo. This work broadens the clinical spectrum of human diseases resulting from reduced CHT activity and highlights the complexity of cholinergic metabolism at the synapse.

Inherited retinal disorders (IRD) represent clinically and genetically heterogeneous diseases. To date, pathogenic variants have been identified in ~260 genes. Albeit that many genes are implicated in IRD, for 30–50% of the cases, the gene defect is unknown. These cases may be explained by novel gene defects, by overlooked structural variants, by variants in intronic, promoter or more distant regulatory regions, and represent synonymous variants of known genes contributing to the dysfunction of the respective proteins. Patients with one subgroup of IRD, namely incomplete congenital stationary night blindness (icCSNB), show a very specific phenotype. The major cause of this condition is the presence of a hemizygous pathogenic variant in CACNA1F. A comprehensive study applying direct Sanger sequencing of the gene‐coding regions, exome and genome sequencing applied to a large cohort of patients with a clinical diagnosis of icCSNB revealed indeed that seven of the 189 CACNA1F‐related cases have intronic and synonymous disease‐causing variants leading to missplicing as validated by minigene approaches. These findings highlight that gene‐locus sequencing may be a very efficient method in detecting disease‐causing variants in clinically well‐characterized patients with a diagnosis of IRD, like icCSNB.

Complete congenital stationary night blindness (cCSNB) is an incurable inherited retinal disorder characterized by an ON-bipolar cell (ON-BC) defect. GRM6 mutations are the third most prevalent cause of cCSNB. The Grm6 −/− mouse model mimics the human phenotype, showing no b-wave in the electroretinogram (ERG) and a loss of mGluR6 and other proteins of the same cascade at the outer plexiform layer (OPL). Our aim was to restore protein localization and function in Grm6 −/− adult mice targeting specifically ON-BCs or the whole retina. METHODS. Adeno-associated virus-encoding Grm6 under two different promoters (GRM6-Grm6 and CAG-Grm6) were injected intravitreally in P15 Grm6 −/− mice. ERG recordings at 2 and 4 months were performed in Grm6 +/+ , untreated and treated Grm6 −/− mice. Similarly, immunolocalization studies were performed on retinal slices before or after treatment using antibodies against mGluR6, TRPM1, GPR179, RGS7, RGS11, Gβ5, and dystrophin. RESULTS. Following treatment, mGluR6 was localized to the dendritic tips of ON-BCs when expressed with either promoter. The relocalization efficiency in mGluR6-transduced retinas at the OPL was 2.5% versus 11% when the GRM6-Grm6 and CAG-Grm6 were used, respectively. Albeit no functional rescue was seen in ERGs, relocalization of TRPM1, GPR179, and Gβ5 was also noted using both constructs. The restoration of the localization of RGS7, RGS11, and dystrophin was more obvious in retinas treated with GRM6-Grm6 than in retinas treated with CAG-Grm6. CONCLUSIONS. Our findings show the potential of treating cCSNB with GRM6 mutations; however, it appears that the transduction rate must be improved to restore visual function.

Complete congenital stationary night blindness (cCSNB) due to mutations in TRPM1 , GRM6 , GPR179 , NYX , or leucine-rich repeat immunoglobulin-like transmembrane domain 3 ( LRIT3 ) is an incurable inherited retinal disorder characterized by an ON-bipolar cell (ON-BC) defect. Since the disease is non-degenerative and stable, treatment could theoretically be administrated at any time in life, making it a promising target for gene therapy. Until now, adeno-associated virus (AAV)-mediated therapies lead to significant functional improvements only in newborn cCSNB mice. Here we aimed to restore protein localization and function in adult Lrit3 −/ − mice. LRIT3 localizes in the outer plexiform layer and is crucial for TRPM1 localization at the dendritic tips of ON-BCs and the electroretinogram (ERG)-b-wave. AAV2-7m8- Lrit3 intravitreal injections were performed targeting either ON-BCs, photoreceptors (PRs), or both. Protein localization of LRIT3 and TRPM1 at the rod-to-rod BC synapse, functional rescue of scotopic responses, and ON-responses detection at the ganglion cell level were achieved in a few mice when ON-BCs alone or both PRs and ON-BCs, were targeted. More importantly, a significant number of treated adult Lrit3 −/− mice revealed an ERG b-wave recovery under scotopic conditions, improved optomotor responses, and on-time ON-responses at the ganglion cell level when PRs were targeted. Functional rescue was maintained for at least 4 months after treatment.

Melanoma-associated retinopathy (MAR) is a rare paraneoplastic retinal disorder usually occurring in the context of metastatic melanoma. Patients present with night blindness, photopsias and a constriction of the visual field. MAR is an auto-immune disorder characterized by the production of autoantibodies targeting retinal proteins, especially autoantibodies reacting to the cation channel TRPM1 produced in melanocytes and ON-bipolar cells. TRPM1 has at least three different isoforms which vary in the N-terminal region of the protein. In this study, we report the case of three new MAR patients presenting different anti-TRPM1 autoantibodies reacting to the three isoforms of TRPM1 with variable binding affinity. Two sera recognized all isoforms of TRPM1, while one recognized only the two longest isoforms upon immunolocalization studies on overexpressing cells. Similarly, the former two sera reacted with all TRPM1 isoforms on western blot, but an immunoprecipitation enrichment step was necessary to detect all isoforms with the latter serum. In contrast, all sera labelled ON-bipolar cells on Tprm1 +/+ but not on Trpm1-/mouse retina as shown by coimmunolocalization. This confirms that the MAR sera specifically detect TRPM1. Most likely, the anti-TRPM1 autoantibodies of different patients vary in affinity and concentration. In addition, the binding of autoantibodies to TRPM1 may be conformation-dependent, with epitopes being inaccessible in some constructs (truncated polypeptides versus full-length TRPM1) or applications (western blotting versus immunohistochemistry).

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Purpose: Myopia is the most common eye disorder, caused by heterogeneous genetic and environmental factors. Rare inherited retinal disorders are often associated with high myopia. Genes implicated in myopia encode proteins involved in eye morphogenesis, extracellular matrix organization, visual perception, circadian rhythms, and retinal signalling. Differentially expressed genes (DEGs) identified in animal models mimicking myopia are helpful in suggesting candidate genes implicated in human myopia. Complete congenital stationary night blindness (cCSNB) in humans and animal models represents an ON‐bipolar cell signal transmission defect and is also associated with high myopia. The purpose of this work was to identify the molecular cause of myopia present in cCSNB. Methods: A whole transcriptome sequencing (RNA‐seq) approach was performed using retina from three mouse lines with cCSNB, Gpr179−/−, Lrit3−/− and Grm6−/− and the expression data compared to data of age‐matched wild‐type littermates (n = 5). DEGs with fold changes of at least 1.2, p‐values of ≤0.01, an expression value of at least 5 transcripts per million reads and appearing in at least two cCSNB mouse lines were investigated in detail. A meta‐analysis was performed by (a) comparing our data with published transcriptome data from purified retinal cells and single cell RNA‐Seq data, (b) pathway analyses, (c) myopia databases and publications concerning their role in normal vision. Several of the DEGs were validated by RT‐qPCR experiments and analysed by western blot and immunolocalization studies. Results: More than 50 DEGs were found in at least two cCSNB mouse lines. Expression of those was found in different retinal cell types. The difference in expression was independent of the number of nuclei present in wild‐type and mutant inner retina. Pathway analysis revealed that mitogen‐activated protein kinase pathways, synaptic signalling, G protein‐coupled receptor ligand binding pathways, and proteins implicated in eye, endoderm and connective tissue development were affected in cCSNB. More than half of the genes were already associated with myopia. Conclusions: Our study reveals DEGs in all retinal cell types of cCSNB models, previously associated with myopia and novel candidates. These studies combined with pathway analyses provide the basis for the development of pharmacological and optical myopia therapies.

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