Clinical Characteristics of POC1B-Associated Retinopathy and Assignment of Pathogenicity to Novel Deep Intronic and Non-Canonical Splice Site Variants

Mutations in POC1B are a rare cause of inherited retinal degeneration. In this study, we present a thorough phenotypic and genotypic characterization of three individuals harboring putatively pathogenic variants in the POC1B gene. All patients displayed a similar, slowly progressive retinopathy (cone dystrophy or cone-rod dystrophy) with normal funduscopy but disrupted outer retinal layers on optical coherence tomography and variable age of onset. Other symptoms were decreased visual acuity and photophobia. Whole genome sequencing revealed a novel homozygous frameshift variant in one patient. Another patient was shown to harbor a novel deep intronic variant in compound heterozygous state with a previously reported canonical splice site variant. The third patient showed a novel nonsense variant and a novel non-canonical splice site variant. We aimed to validate the effect of the deep intronic variant and the non-canonical splice site variant by means of in vitro splice assays. In addition, direct RNA analysis was performed in one patient. Splicing analysis revealed that the non-canonical splice site variant c.561-3T>C leads to exon skipping while the novel deep intronic variant c.1033-327T>A causes pseudoexon activation. Our data expand the genetic landscape of POC1B mutations and confirm the benefit of genome sequencing in combination with downstream functional validation using minigene assays for the analysis of putative splice variants. In addition, we provide clinical multimodal phenotyping of the affected individuals.     

Expanding the Clinical and Genetic Spectrum of RAB28-Related Cone-Rod Dystrophy: Pathogenicity of Novel Variants in Italian Families

The small Ras-related GTPase Rab-28 is highly expressed in photoreceptor cells, where it possibly participates in membrane trafficking. To date, six alterations in the RAB28 gene have been associated with autosomal recessive cone-rod dystrophies. Confirmed variants include splicing variants, missense and nonsense mutations. Here, we present a thorough phenotypical and genotypical characterization of five individuals belonging to four Italian families, constituting the largest cohort of RAB28 patients reported in literature to date. All probands displayed similar clinical phenotype consisting of photophobia, decreased visual acuity, central outer retinal thinning, and impaired color vision. By sequencing the four probands, we identified: a novel homozygous splicing variant; two novel nonsense variants in homozygosis; a novel missense variant in compound heterozygous state with a previously reported nonsense variant. Exhaustive molecular dynamics simulations of the missense variant p.(Thr26Asn) in both its active and inactive states revealed an allosteric structural mechanism that impairs the binding of Mg²⁺, thus decreasing the affinity for GTP. The impaired GTP-GDP exchange ultimately locks Rab-28 in a GDP-bound inactive state. The loss-of-function mutation p.(Thr26Asn) was present in a compound heterozygosis with the nonsense variant p.(Arg137*), which does not cause mRNA-mediated decay, but is rather likely degraded due to its incomplete folding. The frameshift p.(Thr26Valfs4*) and nonsense p.(Leu13*) and p.(Trp107*) variants, if translated, would lack several key structural components necessary for the correct functioning of the encoded protein.     

Autosomal Dominant Gyrate Atrophy-Like Choroidal Dystrophy Revisited: 45 Years Follow-Up and Association with a Novel C1QTNF5 Missense Variant

We present a long-term follow-up in autosomal dominant gyrate atrophy-like choroidal dystrophy (adGALCD) and propose a possible genotype/phenotype correlation. Ophthalmic examination of six patients from two families revealed confluent areas of choroidal atrophy resembling gyrate atrophy, starting in the second decade of life. Progression continued centrally, reaching the fovea at about 60 years of age. Subretinal deposits, retinal pigmentation or choroidal neovascularization as seen in late-onset retinal degeneration (LORD) were not observed. Whole genome sequencing revealed a novel missense variant in the C1QTNF5 gene (p.(Q180E)) which was found in heterozygous state in all affected subjects. Haplotype analysis showed that this variant found in both families is identical by descent. Three-dimensional modeling of the possible supramolecular assemblies of C1QTNF5 revealed that the p.(Q180E) variant led to the destabilization of protein tertiary and quaternary structures, affecting both the stability of the single protomer and the entire globular head, thus exerting detrimental effects on the formation of C1QTNF5 trimeric globular domains and their interaction. In conclusion, we propose that the p.(Q180E) variant causes a specific phenotype, adGALCD, that differs in multiple clinical aspects from LORD. Disruption of optimal cell-adhesion mechanisms is expected when analyzing the effects of the point mutation at the protein level.     

Molecular and Clinical Implications of Variant Repeats in Myotonic Dystrophy Type 1

Myotonic dystrophy type 1 (DM1) is one of the most variable monogenic diseases at phenotypic, genetic, and epigenetic level. The disease is multi-systemic with the age at onset ranging from birth to late age. The underlying mutation is an unstable expansion of CTG repeats in the DMPK gene, varying in size from 50 to >1000 repeats. Generally, large expansions are associated with an earlier age at onset. Additionally, the most severe, congenital DM1 form is typically associated with local DNA methylation. Genetic variability of DM1 mutation is further increased by its structural variations due to presence of other repeats (e.g., CCG, CTC, CAG). These variant repeats or repeat interruptions seem to confer an additional level of epigenetic variability since local DNA methylation is frequently associated with variant CCG repeats independently of the expansion size. The effect of repeat interruptions on DM1 molecular pathogenesis is not investigated enough. Studies on patients indicate their stabilizing effect on DMPK expansions because no congenital cases were described in patients with repeat interruptions, and the age at onset is frequently later than expected. Here, we review the clinical relevance of repeat interruptions in DM1 and genetic and epigenetic characteristics of interrupted DMPK expansions based on patient studies.     

CRB1-Related Retinal Dystrophies in a Cohort of 50 Patients: A Reappraisal in the Light of Specific Müller Cell and Photoreceptor CRB1 Isoforms

Pathogenic variants in CRB1 lead to diverse recessive retinal disorders from severe Leber congenital amaurosis to isolated macular dystrophy. Until recently, no clear phenotype-genotype correlation and no appropriate mouse models existed. Herein, we reappraise the phenotype-genotype correlation of 50 patients with regards to the recently identified CRB1 isoforms: a canonical long isoform A localized in Müller cells (12 exons) and a short isoform B predominant in photoreceptors (7 exons). Twenty-eight patients with early onset retinal dystrophy (EORD) consistently had a severe Müller impairment, with variable impact on the photoreceptors, regardless of isoform B expression. Among them, two patients expressing wild type isoform B carried one variant in exon 12, which specifically damaged intracellular protein interactions in Müller cells. Thirteen retinitis pigmentosa patients had mainly missense variants in laminin G-like domains and expressed at least 50% of isoform A. Eight patients with the c.498_506del variant had macular dystrophy. In one family homozygous for the c.1562C>T variant, the brother had EORD and the sister macular dystrophy. In contrast with the mouse model, these data highlight the key role of Müller cells in the severity of CRB1-related dystrophies in humans, which should be taken into consideration for future clinical trials.     

Inherited Retinal Diseases Due to RPE65 Variants: From Genetic Diagnostic Management to Therapy

Inherited retinal diseases (IRDs) are a heterogeneous group of conditions that include retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA) and early-onset severe retinal dystrophy (EO[S]RD), which differ in severity and age of onset. IRDs are caused by mutations in >250 genes. Variants in the RPE65 gene account for 0.6–6% of RP and 3–16% of LCA/EORD cases. Voretigene neparvovec is a gene therapy approved for the treatment of patients with an autosomal recessive retinal dystrophy due to confirmed biallelic RPE65 variants (RPE65-IRDs). Therefore, the accurate molecular diagnosis of RPE65-IRDs is crucial to identify ‘actionable’ genotypes—i.e., genotypes that may benefit from the treatment—and is an integral part of patient management. To date, hundreds of RPE65 variants have been identified, some of which are classified as pathogenic or likely pathogenic, while the significance of others is yet to be established. In this review, we provide an overview of the genetic diagnostic workup needed to select patients that could be eligible for voretigene neparvovec treatment. Careful clinical characterization of patients by multidisciplinary teams of experts, combined with the availability of next-generation sequencing approaches, can accelerate patients’ access to available therapeutic options.     

Polymorphism of the Flap Endonuclease 1 Gene in Keratoconus and Fuchs Endothelial Corneal Dystrophy

Oxidative stress is implicated in the pathogenesis of many diseases, including serious ocular diseases, keratoconus (KC) and Fuchs endothelial corneal dystrophy (FECD). Flap endonuclease 1 (FEN1) plays an important role in the repair of oxidative DNA damage in the base excision repair pathway. We determined the association between two single nucleotide polymorphisms (SNPs), c.–441G>A (rs174538) and g.61564299G>T (rs4246215), in the FEN1 gene and the occurrence of KC and FECD. This study involved 279 patients with KC, 225 patients with FECD and 322 control individuals. Polymerase chain reaction (PCR) and length polymorphism restriction fragment analysis (RFLP) were applied. The T/T genotype of the g.61564299G>T polymorphism was associated with an increased occurrence of KC and FECD. There was no association between the c.–441G>A polymorphism and either disease. However, the GG haplotype of both polymorphisms was observed more frequently and the GT haplotype less frequently in the KC group than the control. The AG haplotype was associated with increased FECD occurrence. Our findings suggest that the g.61564299G>T and c.–441G>A polymorphisms in the FEN1 gene may modulate the risk of keratoconus and Fuchs endothelial corneal dystrophy.     

Targeting Myotonic Dystrophy Type 1 with Metformin

Myotonic dystrophy type 1 (DM1) is a multisystemic disorder of genetic origin. Progressive muscular weakness, atrophy and myotonia are its most prominent neuromuscular features, while additional clinical manifestations in multiple organs are also common. Overall, DM1 features resemble accelerated aging. There is currently no cure or specific treatment for myotonic dystrophy patients. However, in recent years a great effort has been made to identify potential new therapeutic strategies for DM1 patients. Metformin is a biguanide antidiabetic drug, with potential to delay aging at cellular and organismal levels. In DM1, different studies revealed that metformin rescues multiple phenotypes of the disease. This review provides an overview of recent findings describing metformin as a novel therapy to combat DM1 and their link with aging.     

Deciphering the Complex Molecular Pathogenesis of Myotonic Dystrophy Type 1 through Omics Studies

Omics studies are crucial to improve our understanding of myotonic dystrophy type 1 (DM1), the most common muscular dystrophy in adults. Employing tissue samples and cell lines derived from patients and animal models, omics approaches have revealed the myriad alterations in gene and microRNA expression, alternative splicing, 3′ polyadenylation, CpG methylation, and proteins levels, among others, that contribute to this complex multisystem disease. In addition, omics characterization of drug candidate treatment experiments provides crucial insight into the degree of therapeutic rescue and off-target effects that can be achieved. Finally, several innovative technologies such as single-cell sequencing and artificial intelligence will have a significant impact on future DM1 research.     

Allosteric Modulation of GSK-3β as a New Therapeutic Approach in Limb Girdle Muscular Dystrophy R1 Calpain 3-Related

Limb-girdle muscular dystrophy R1 calpain 3-related (LGMDR1) is an autosomal recessive muscular dystrophy produced by mutations in the CAPN3 gene. It is a rare disease and there is no cure or treatment for the disease while the pathophysiological mechanism by which the absence of calpain 3 provokes the dystrophy in muscles is not clear. However, key proteins implicated in Wnt and mTOR signaling pathways, which regulate muscle homeostasis, showed a considerable reduction in their expression and in their phosphorylation in LGMDR1 patients’ muscles. Finally, the administration of tideglusib and VP0.7, ATP non-competitive inhibitors of glycogen synthase kinase 3β (GSK-3β), restore the expression and phosphorylation of these proteins in LGMDR1 cells, opening the possibility of their use as therapeutic options.     

Therapeutic Application of Extracellular Vesicles-Capsulated Adeno-Associated Virus Vector via nSMase2/Smpd3, Satellite,and Immune Cells in Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is caused by loss-of-function mutations in the dystrophin gene on chromosome Xp21. Disruption of the dystrophin–glycoprotein complex (DGC) on the cell membrane causes cytosolic Ca²⁺ influx, resulting in protease activation, mitochondrial dysfunction, and progressive myofiber degeneration, leading to muscle wasting and fragility. In addition to the function of dystrophin in the structural integrity of myofibers, a novel function of asymmetric cell division in muscular stem cells (satellite cells) has been reported. Therefore, it has been suggested that myofiber instability may not be the only cause of dystrophic degeneration, but rather that the phenotype might be caused by multiple factors, including stem cell and myofiber functions. Furthermore, it has been focused functional regulation of satellite cells by intracellular communication of extracellular vesicles (EVs) in DMD pathology. Recently, a novel molecular mechanism of DMD pathogenesis—circulating RNA molecules—has been revealed through the study of target pathways modulated by the Neutral sphingomyelinase2/Neutral sphingomyelinase3 (nSMase2/Smpd3) protein. In addition, adeno-associated virus (AAV) has been clinically applied for DMD therapy owing to the safety and long-term expression of transduction genes. Furthermore, the EV-capsulated AAV vector (EV-AAV) has been shown to be a useful tool for the intervention of DMD, because of the high efficacy of the transgene and avoidance of neutralizing antibodies. Thus, we review application of AAV and EV-AAV vectors for DMD as novel therapeutic strategy.     

Skeletal Ryanodine Receptors Are Involved in Impaired Myogenic Differentiation in Duchenne Muscular Dystrophy Patients

Duchenne muscular dystrophy (DMD) is characterized by progressive muscle wasting following repeated muscle damage and inadequate regeneration. Impaired myogenesis and differentiation play a major role in DMD as well as intracellular calcium (Ca²⁺) mishandling. Ca²⁺ release from the sarcoplasmic reticulum is mostly mediated by the type 1 ryanodine receptor (RYR1) that is required for skeletal muscle differentiation in animals. The study objective was to determine whether altered RYR1-mediated Ca²⁺ release contributes to myogenic differentiation impairment in DMD patients. The comparison of primary cultured myoblasts from six boys with DMD and five healthy controls highlighted delayed myoblast differentiation in DMD. Silencing RYR1 expression using specific si-RNA in a healthy control induced a similar delayed differentiation. In DMD myotubes, resting intracellular Ca²⁺ concentration was increased, but RYR1-mediated Ca²⁺ release was not changed compared with control myotubes. Incubation with the RYR-calstabin interaction stabilizer S107 decreased resting Ca²⁺ concentration in DMD myotubes to control values and improved calstabin1 binding to the RYR1 complex. S107 also improved myogenic differentiation in DMD. Furthermore, intracellular Ca²⁺ concentration was correlated with endomysial fibrosis, which is the only myopathologic parameter associated with poor motor outcome in patients with DMD. This suggested a potential relationship between RYR1 dysfunction and motor impairment. Our study highlights RYR1-mediated Ca²⁺ leakage in human DMD myotubes and its key role in myogenic differentiation impairment. RYR1 stabilization may be an interesting adjunctive therapeutic strategy in DMD.     

Complex Transposon Insertion as a Novel Cause of Pompe Disease

Pompe disease (OMIM#232300) is an autosomal recessive lysosomal storage disorder caused by mutations in the GAA gene. According to public mutation databases, more than 679 pathogenic variants have been described in GAA, none of which are associated with mobile genetic elements. In this article, we report a novel molecular genetic cause of Pompe disease, which could be hardly detected using routine molecular genetic analysis. Whole genome sequencing followed by comprehensive functional analysis allowed us to discover and characterize a complex mobile genetic element insertion deep in the intron 15 of the GAA gene in a patient with infantile onset Pompe disease.     

A Dystrophin Exon-52 Deleted Miniature Pig Model of Duchenne Muscular Dystrophy and Evaluation of Exon Skipping

Duchenne muscular dystrophy (DMD) is a lethal X-linked recessive disorder caused by mutations in the DMD gene and the subsequent lack of dystrophin protein. Recently, phosphorodiamidate morpholino oligomer (PMO)-antisense oligonucleotides (ASOs) targeting exon 51 or 53 to reestablish the DMD reading frame have received regulatory approval as commercially available drugs. However, their applicability and efficacy remain limited to particular patients. Large animal models and exon skipping evaluation are essential to facilitate ASO development together with a deeper understanding of dystrophinopathies. Using recombinant adeno-associated virus-mediated gene targeting and somatic cell nuclear transfer, we generated a Yucatan miniature pig model of DMD with an exon 52 deletion mutation equivalent to one of the most common mutations seen in patients. Exon 52-deleted mRNA expression and dystrophin deficiency were confirmed in the skeletal and cardiac muscles of DMD pigs. Accordingly, dystrophin-associated proteins failed to be recruited to the sarcolemma. The DMD pigs manifested early disease onset with severe bodywide skeletal muscle degeneration and with poor growth accompanied by a physical abnormality, but with no obvious cardiac phenotype. We also demonstrated that in primary DMD pig skeletal muscle cells, the genetically engineered exon-52 deleted pig DMD gene enables the evaluation of exon 51 or 53 skipping with PMO and its advanced technology, peptide-conjugated PMO. The results show that the DMD pigs developed here can be an appropriate large animal model for evaluating in vivo exon skipping efficacy.     

Identification of Specific Variations in a Non-Motile Strain of Cyanobacterium Synechocystis sp. PCC 6803 Originated from ATCC 27184 by Whole Genome Resequencing

Cyanobacterium Synechocystis sp. PCC 6803 is a widely used model organism in basic research and biofuel biotechnology application. Here, we report the genomic sequence of chromosome and seven plasmids of a glucose-tolerant, non-motile strain originated from ATCC 27184, GT-G, in use at Guangzhou. Through high-throughput genome re-sequencing and verification by Sanger sequencing, eight novel variants were identified in its chromosome and plasmids. The eight novel variants, especially the five non-silent mutations might have interesting effects on the phenotype of GT-G strains, for example the truncated Sll1895 and Slr0322 protein. These resequencing data provide background information for further research and application based on the GT-G strain and also provide evidence to study the evolution and divergence of Synechocystis 6803 globally.     

Lamin-Related Congenital Muscular Dystrophy Alters Mechanical Signaling and Skeletal Muscle Growth

Laminopathies are a clinically heterogeneous group of disorders caused by mutations in the LMNA gene, which encodes the nuclear envelope proteins lamins A and C. The most frequent diseases associated with LMNA mutations are characterized by skeletal and cardiac involvement, and include autosomal dominant Emery–Dreifuss muscular dystrophy (EDMD), limb-girdle muscular dystrophy type 1B, and LMNA-related congenital muscular dystrophy (LMNA-CMD). Although the exact pathophysiological mechanisms responsible for LMNA-CMD are not yet understood, severe contracture and muscle atrophy suggest that mutations may impair skeletal muscle growth. Using human muscle stem cells (MuSCs) carrying LMNA-CMD mutations, we observe impaired myogenic fusion with disorganized cadherin/β catenin adhesion complexes. We show that skeletal muscle from Lmna-CMD mice is unable to hypertrophy in response to functional overload, due to defective fusion of activated MuSCs, defective protein synthesis and defective remodeling of the neuromuscular junction. Moreover, stretched myotubes and overloaded muscle fibers with LMNA-CMD mutations display aberrant mechanical regulation of the yes-associated protein (YAP). We also observe defects in MuSC activation and YAP signaling in muscle biopsies from LMNA-CMD patients. These phenotypes are not recapitulated in closely related but less severe EDMD models. In conclusion, combining studies in vitro, in vivo, and patient samples, we find that LMNA-CMD mutations interfere with mechanosignaling pathways in skeletal muscle, implicating A-type lamins in the regulation of skeletal muscle growth.