The new missense G376V-TDP-43 variant induces late-onset distal myopathy but not amyotrophic lateral sclerosis.

TDP-43-positive inclusions in neurons are a hallmark of several neurodegenerative diseases including familial amyotrophic lateral sclerosis (fALS) caused by pathogenic TARDBP variants as well as more common non-Mendelian sporadic ALS (sALS). Here we report a G376V-TDP-43 missense variant in the C-terminal prion-like domain of the protein in two French families affected by an autosomal dominant myopathy but not fulfilling diagnostic criteria for ALS. Patients from both families presented with progressive weakness and atrophy of distal muscles, starting in their 5th-7th decade. Muscle biopsies revealed a degenerative myopathy characterized by accumulation of rimmed (autophagic) vacuoles, disruption of sarcomere integrity and severe myofibrillar disorganization. The G376 V variant altered a highly conserved amino acid residue and was absent in databases on human genome variation. Variant pathogenicity was supported by in silico analyses and functional studies. The G376 V mutant increased the formation of cytoplasmic TDP-43 condensates in cell culture models, promoted assembly into high molecular weight oligomers and aggregates in vitro, and altered morphology of TDP-43 condensates arising from phase separation. Moreover, the variant led to the formation of cytoplasmic TDP-43 condensates in patient-derived myoblasts and induced abnormal mRNA splicing in patient muscle tissue. The identification of individuals with TDP-43-related myopathy but not ALS implies that TARDBP missense variants may have more pleiotropic effects than previously anticipated and support a primary role for TDP-43 in skeletal muscle pathophysiology. We propose to include TARDBP screening in the genetic work-up of patients with late-onset distal myopathy. Further research is warranted to examine the precise pathogenic mechanisms of TARDBP variants causing either a neurodegenerative or myopathic phenotype.

Biallelic variants in HPDL cause pure and complicated hereditary spastic paraplegia.

Human 4-hydroxyphenylpyruvate dioxygenase-like (HPDL) is a putative iron-containing non-heme oxygenase of unknown specificity and biological significance. We report 25 families containing 34 individuals with neurological disease associated with biallelic HPDL variants. Phenotypes ranged from juvenile-onset pure hereditary spastic paraplegia to infantile-onset spasticity and global developmental delays, sometimes complicated by episodes of neurological and respiratory decompensation. Variants included bona fide pathogenic truncating changes, although most were missense substitutions. Functionality of variants could not be determined directly as the enzymatic specificity of HPDL is unknown; however, when HPDL missense substitutions were introduced into 4-hydroxyphenylpyruvate dioxygenase (HPPD, an HPDL orthologue), they impaired the ability of HPPD to convert 4-hydroxyphenylpyruvate into homogentisate. Moreover, three additional sets of experiments provided evidence for a role of HPDL in the nervous system and further supported its link to neurological disease: (i) HPDL was expressed in the nervous system and expression increased during neural differentiation; (ii) knockdown of zebrafish hpdl led to abnormal motor behaviour, replicating aspects of the human disease; and (iii) HPDL localized to mitochondria, consistent with mitochondrial disease that is often associated with neurological manifestations. Our findings suggest that biallelic HPDL variants cause a syndrome varying from juvenile-onset pure hereditary spastic paraplegia to infantile-onset spastic tetraplegia associated with global developmental delays.

PRDM12 Is Required for Initiation of the Nociceptive Neuron Lineage during Neurogenesis.

The sensation of pain is essential for the preservation of the functional integrity of the body. However, the key molecular regulators necessary for the initiation of the development of pain-sensing neurons have remained largely unknown. Here, we report that, in mice, inactivation of the transcriptional regulator PRDM12, which is essential for pain perception in humans, results in a complete absence of the nociceptive lineage, while proprioceptive and touch-sensitive neurons remain. Mechanistically, our data reveal that PRDM12 is required for initiation of neurogenesis and activation of a cascade of downstream pro-neuronal transcription factors, including NEUROD1, BRN3A, and ISL1, in the nociceptive lineage while it represses alternative fates other than nociceptors in progenitor cells. Our results thus demonstrate that PRDM12 is necessary for the generation of the entire lineage of pain-initiating neurons.

MPV17 mutations in juvenile- and adult-onset axonal sensorimotor polyneuropathy.

MPV17 encodes a putative channel-forming protein of the inner mitochondrial membrane and is involved in mitochondrial deoxynucleotide homeostasis. MPV17 mutations were first reported in patients with Navajo neurohepatopathy, an autosomal recessive mitochondrial DNA depletion syndrome, characterized by early-onset liver failure, failure to thrive as well as central and peripheral neurological involvement. Recently, two patients with juvenile-onset peripheral sensorimotor neuropathy associated with an MVP17 c.122G>A (p.Arg41Gln) variant have been reported. Here, we describe five additional patients from two unrelated families with sensorimotor axonal neuropathy without hepatocerebral affection caused by homozygous MPV17 variants. Patients of the first family carried the known c.122G>A variant and affected individuals of the second family had a novel c.376-9T>G near-splice variant, which was shown to result in an in-frame deletion of 11 amino acids. This report provides further evidence that MPV17 mutations should be considered in patients with pure, non-syndromic axonal neuropathy.

Loss of tubulin deglutamylase CCP1 causes infantile-onset neurodegeneration.

A set of glutamylases and deglutamylases controls levels of tubulin polyglutamylation, a prominent post-translational modification of neuronal microtubules. Defective tubulin polyglutamylation was first linked to neurodegeneration in the Purkinje cell degeneration (pcd) mouse, which lacks deglutamylase CCP1, displays massive cerebellar atrophy, and accumulates abnormally glutamylated tubulin in degenerating neurons. We found biallelic rare and damaging variants in the gene encoding CCP1 in 13 individuals with infantile-onset neurodegeneration and confirmed the absence of functional CCP1 along with dysregulated tubulin polyglutamylation. The human disease mainly affected the cerebellum, spinal motor neurons, and peripheral nerves. We also demonstrate previously unrecognized peripheral nerve and spinal motor neuron degeneration in pcd mice, which thus recapitulated key features of the human disease. Our findings link human neurodegeneration to tubulin polyglutamylation, entailing this post-translational modification as a potential target for drug development for neurodegenerative disorders.

Mutations in INPP5K, Encoding a Phosphoinositide 5-Phosphatase, Cause Congenital Muscular Dystrophy with Cataracts and Mild Cognitive Impairment.

Phosphoinositides are small phospholipids that control diverse cellular downstream signaling events. Their spatial and temporal availability is tightly regulated by a set of specific lipid kinases and phosphatases. Congenital muscular dystrophies are hereditary disorders characterized by hypotonia and weakness from birth with variable eye and central nervous system involvement. In individuals exhibiting congenital muscular dystrophy, early-onset cataracts, and mild intellectual disability but normal cranial magnetic resonance imaging, we identified bi-allelic mutations in INPP5K, encoding inositol polyphosphate-5-phosphatase K. Mutations impaired phosphatase activity toward the phosphoinositide phosphatidylinositol (4,5)-bisphosphate or altered the subcellular localization of INPP5K. Downregulation of INPP5K orthologs in zebrafish embryos disrupted muscle fiber morphology and resulted in abnormal eye development. These data link congenital muscular dystrophies to defective phosphoinositide 5-phosphatase activity that is becoming increasingly recognized for its role in mediating pivotal cellular mechanisms contributing to disease.

Targeted HIV-1 Latency Reversal Using CRISPR/Cas9-Derived Transcriptional Activator Systems.

CRISPR/Cas9 technology is currently considered the most advanced tool for targeted genome engineering. Its sequence-dependent specificity has been explored for locus-directed transcriptional modulation. Such modulation, in particular transcriptional activation, has been proposed as key approach to overcome silencing of dormant HIV provirus in latently infected cellular reservoirs. Currently available agents for provirus activation, so-called latency reversing agents (LRAs), act indirectly through cellular pathways to induce viral transcription. However, their clinical performance remains suboptimal, possibly because reservoirs have diverse cellular identities and/or proviral DNA is intractable to the induced pathways. We have explored two CRISPR/Cas9-derived activator systems as targeted approaches to induce dormant HIV-1 proviral DNA. These systems recruit multiple transcriptional activation domains to the HIV 5' long terminal repeat (LTR), for which we have identified an optimal target region within the LTR U3 sequence. Using this target region, we demonstrate transcriptional activation of proviral genomes via the synergistic activation mediator complex in various in culture model systems for HIV latency. Observed levels of induction are comparable or indeed higher than treatment with established LRAs. Importantly, activation is complete, leading to production of infective viral particles. Our data demonstrate that CRISPR/Cas9-derived technologies can be applied to counteract HIV latency and may therefore represent promising novel approaches in the quest for HIV elimination.

Transcriptional regulator PRDM12 is essential for human pain perception.

Pain perception has evolved as a warning mechanism to alert organisms to tissue damage and dangerous environments. In humans, however, undesirable, excessive or chronic pain is a common and major societal burden for which available medical treatments are currently suboptimal. New therapeutic options have recently been derived from studies of individuals with congenital insensitivity to pain (CIP). Here we identified 10 different homozygous mutations in PRDM12 (encoding PRDI-BF1 and RIZ homology domain-containing protein 12) in subjects with CIP from 11 families. Prdm proteins are a family of epigenetic regulators that control neural specification and neurogenesis. We determined that Prdm12 is expressed in nociceptors and their progenitors and participates in the development of sensory neurons in Xenopus embryos. Moreover, CIP-associated mutants abrogate the histone-modifying potential associated with wild-type Prdm12. Prdm12 emerges as a key factor in the orchestration of sensory neurogenesis and may hold promise as a target for new pain therapeutics.

A 3'-UTR mutation creates a microRNA target site in the GFPT1 gene of patients with congenital myasthenic syndrome.

Mutations in the gene encoding glutamine-fructose-6-phosphate transaminase 1 (GFPT1) cause the neuromuscular disorder limb-girdle congenital myasthenic syndrome (LG-CMS). One recurrent GFPT1 mutation detected in LG-CMS patients is a c.*22C>A transversion in the 3'-untranslated region (UTR). Because this variant does not alter the GFPT1 open reading frame, its pathogenic relevance has not yet been established. We found that GFPT1 protein levels were reduced in myoblast cells of the patients carrying this variant. In silico algorithms predicted that the mutation creates a microRNA target site for miR-206*. Investigation of the expression of this so far unrecognized microRNA confirmed that miR-206* (like its counterpart miR-206) is abundant in skeletal muscle. MiR-206* efficiently reduced the expression of reporter constructs containing the mutated 3'-UTR while no such effect was observed with reporter constructs containing the wild-type 3'-UTR or when a specific anti-miR-206* inhibitor was added. Moreover, anti-miR-206* inhibitor treatment substantially rescued GFPT1 expression levels in patient-derived myoblasts. Our data demonstrate that the c.*22C>A mutation in the GFPT1 gene leads to illegitimate binding of microRNA resulting in reduced protein expression. We confirm that c.*22C>A is a causative mutation and suggest that formation of microRNA target sites might be a relevant pathomechanism in Mendelian disorders. Variants in the 3'-UTRs should be considered in genetic diagnostic procedures.

Salbutamol-responsive limb-girdle congenital myasthenic syndrome due to a novel missense mutation and heteroallelic deletion in MUSK.

Congenital myasthenic syndromes (CMS) are clinically and genetically heterogeneous disorders characterized by a neuromuscular transmission defect. In recent years, causative mutations have been identified in atleast 15 genes encoding proteins of the neuromuscular junction. Mutations in MUSK are known as a very rare genetic cause of CMS and have been described in only three families, world-wide. Consequently, the knowledge about efficient drug therapy is very limited. We identified a novel missense mutation (p.Asp38Glu) heteroallelic to a genomic deletion affecting exons 2-3 of MUSK as cause of a limb-girdle CMS in two brothers of Turkish origin. Clinical symptoms included fatigable limb weakness from early childhood on. Upon diagnosis of a MUSK-related CMS at the age of 16 and 13years, respectively, treatment with salbutamol was initiated leading to an impressive improvement of clinical symptoms, while treatment with esterase inhibitors did not show any benefit. Our findings highlight the importance of a molecular diagnosis in CMS and demonstrate considerable similarities between patients with MUSK and DOK7-related CMS in terms of clinical phenotype and treatment options.

Highly significant antiviral activity of HIV-1 LTR-specific tre-recombinase in humanized mice.

Stable integration of HIV proviral DNA into host cell chromosomes, a hallmark and essential feature of the retroviral life cycle, establishes the infection permanently. Current antiretroviral combination drug therapy cannot cure HIV infection. However, expressing an engineered HIV-1 long terminal repeat (LTR) site-specific recombinase (Tre), shown to excise integrated proviral DNA in vitro, may provide a novel and highly promising antiviral strategy. We report here the conditional expression of Tre-recombinase from an advanced lentiviral self-inactivation (SIN) vector in HIV-infected cells. We demonstrate faithful transgene expression, resulting in accurate provirus excision in the absence of cytopathic effects. Moreover, pronounced Tre-mediated antiviral effects are demonstrated in vivo, particularly in humanized Rag2⁻/⁻γc⁻/⁻ mice engrafted with either Tre-transduced primary CD4⁺ T cells, or Tre-transduced CD34⁺ hematopoietic stem and progenitor cells (HSC). Taken together, our data support the use of Tre-recombinase in novel therapy strategies aiming to provide a cure for HIV.

Long-term preservation of cardiac structure and function after adeno-associated virus serotype 9-mediated microdystrophin gene transfer in mdx mice.

Dystrophin plays an important role in muscle contraction, linking the intracellular cytoskeleton to the extracellular matrix. Mutations of the dystrophin gene leading to a complete loss of the protein cause Duchenne muscular dystrophy (DMD), frequently associated with severe cardiomyopathy. Early clinical trials in DMD using gene transfer to skeletal muscle are underway, but gene transfer to dystrophic cardiac muscle has not yet been tested in humans. The aim of this study was to develop an optimized protocol for cardiac gene therapy in the mouse model of dystrophin deficiency (mdx), using a cardiac promoter for expression of a microdystrophin (µDys) transgene packaged into an adeno-associated virus serotype 9 vector (AAV9). In this study adult mdx mice were intravenously injected with 1×10(12) genomic particles of AAV9 vectors carrying a cDNA encoding µDys under the control of either a ubiquitously active cytomegalovirus (CMV) promoter or a cardiac-specific CMV-enhanced myosin light chain (MLC0.26) promoter. After 10 months, both AAV9 vectors led to sustained µDys expression in cardiac muscle, but the MLC promoter conferred about 4-fold higher protein levels. AAV9-CMV-MLC0.26-µDys resulted in significant protection of cardiac morphology and function as assessed by histopathology, echocardiography, and left ventricular catheterization. In conclusion, we established an AAV9-mediated gene transfer approach for efficient and specific long-term µDys expression in the hearts of mdx mice, resulting in a sustained therapeutic effect. Thus, this approach might be a basis for further translation into a treatment strategy for DMD-associated cardiomyopathy.

Genomic integration of adenoviral gene transfer vectors following transduction of fertilized mouse oocytes.

Adenoviral vectors (AdV) are popular tools to deliver foreign genes into a wide range of cells. They have also been used in clinical gene therapy trials. Studies on AdV-mediated gene transfer to mammalian oocytes and transmission through the germ line have been reported controversially. In the present study we investigated whether AdV sequences integrate into the mouse genome by microinjecting AdV into the perivitelline space of fertilized oocytes. We applied a newly developed PCR technique (HiLo-PCR) for identification of chromosomal junctions next to the integrated AdV. We demonstrate that mouse oocytes can be transduced by different recombinant adenoviral vectors (first generation and gutless). In one transgenic mouse line using the first generation adenoviral vector, the genome has integrated into a highly repetitive cluster located on the Y chromosome. While the transgene (GFP) was expressed in early embryos, no expression was detected in adult transgenic mice. The use of gutless AdV resulted in expression of the transgene, albeit the vector was not transmitted to progeny. These results indicate that under optimized conditions fertilized mouse oocytes are transduced by AdV and give rise to transgenic founder animals. Therefore, adequate precautions should be taken in gene therapy protocols of reproductive patients since transduction of oocytes or early embryos and subsequent chromosomal integration cannot be ruled out entirely.

Clinical and neuropathological findings in patients with TACO1 mutations.

We have recently identified mutations in the translation activator of cytochrome c oxidase 1 (TACO1) gene, leading to cytochrome c oxidase (COX) deficiency. Here, we report the clinical and neuroimaging findings of five members of a big consanguinous family homozygous for c.472insC in TACO1. All 5 patients had an uneventful early childhood and a subtle onset, slowly progressive cognitive dysfunction, dystonia or visual impairment between ages 4 and 16years. Affected girls had a milder phenotype and preserved ambulation into the late twenties. Brain MRI revealed bilateral, symmetric lesions of the basal ganglia in all affected family members, but less prominent in girls. TACO1 analysis showed no mutations in 17 patients with juvenile-onset Leigh syndrome and isolated COX or combined respiratory chain deficiency, indicating that TACO1 mutations are a rare cause of Leigh syndrome.

Intragenic deletion of TRIM32 in compound heterozygotes with sarcotubular myopathy/LGMD2H.

In 2005 the commonality of sarcotubular myopathy (STM) and limb girdle muscular dystrophy type 2H (LGMD2H) was demonstrated, as both are caused by the p D487N missense mutation in TRIM32 originally found in the Manitoba Hutterite population. Recently, three novel homozygous TRIM32 mutations have been described in LGMD patients. Here we describe a three generation Swedish family clinically presenting with limb girdle muscular weakness and histological features of a microvacuolar myopathy. The two index patients were compound heterozygotes for a frameshift mutation in TRIM32 (c.1560delC ) and a 30 kb intragenic deletion, encompassing parts of intron 1 and the entire exon 2 of TRIM32. In these patients, no full-length or truncated TRIM32 could be detected. Interestingly, heterozygous family members carrying only one mutation showed mild clinical symptoms and vacuolar changes in muscle. In our family, the phenotype encompasses additionally a mild demyelinating polyneuropathic syndrome. Thus STM and LGMD2H are the result of loss of function mutations that can be either deletions or missense mutations.

Efficient and fast functional screening of microdystrophin constructs in vivo and in vitro for therapy of duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is an X-linked, lethal genetic disorder affecting the skeletal muscle compartment, and is caused by mutation(s) in the dystrophin gene. Gene delivery of microdystrophin constructs using adeno-associated virus (AAV) and antisense-mediated exon skipping restoring the genetic reading frame are two of the most promising therapeutic strategies for DMD. Both approaches use microdystrophin proteins either directly as a desired construct for gene delivery, using the capacity-limited AAV vectors, or as the therapeutic outcome of gene splicing. Although functionality of the resulting artificial dystrophin proteins can be predicted in silico, experimental evidence usually obtained in transgenic mice is required before human trials. However, the enormous number of potential constructs makes screening assays for dystrophin protein function in vitro and in vivo highly desirable. Here we present data showing that functionality of microdystrophins can be assessed using relatively simple and fast techniques.

A newly identified chromosomal microdeletion of the rapsyn gene causes a congenital myasthenic syndrome.

The objective is mutation analysis of the RAPSN gene in a patient with sporadic congenital myasthenic syndrome (CMS). Mutations in various genes encoding proteins expressed at the neuromuscular junction may cause CMS. Most mutations affect the epsilon subunit gene of the acetylcholine receptor (AChR) leading to endplate AChR deficiency. Recently, mutations in the RAPSN gene have been identified in several CMS patients with AChR deficiency. In most patients, RAPSN N88K was identified, either homozygously or heteroallelic to a second missense mutation. A sporadic CMS patient from Germany was analyzed for RAPSN mutations by RFLP, long-range PCR and sequence analysis. Clinically, the patient presents with an early onset CMS, associated with arthrogryposis multiplex congenita, recurrent episodes of respiratory insufficiency provoked by infections, and a moderate general weakness, responsive to anticholinesterase treatment. The mutation RAPSN N88K was found heterozygously to a large deletion of about 4.5 kb disrupting the RAPSN gene. Interestingly, an Alu-mediated unequal homologous recombination may have caused the deletion. We hypothesize that numerous interspersed Alu elements may predispose the RAPSN locus for genetic rearrangements.

Expression of dystrophin driven by the 1.35-kb MCK promoter ameliorates muscular dystrophy in fast, but not in slow muscles of transgenic mdx mice.

Successful gene therapy of Duchenne muscular dystrophy may require the lifelong expression of a therapeutic gene in all affected muscles. The most promising gene delivery vehicles, viral vectors, suffer from several limitations, including immunogenicity, loss of therapeutic gene expression, and a limited packaging capacity. Therefore, various efforts were previously undertaken to use small therapeutic genes and to place them under the control of a strong and muscle-specific promoter. Here we report the effects of a minidystrophin (6.3 kb) under the control of a short muscle-specific promoter (MCK 1.35 kb) over most of the lifetime (4-20 months) of a transgenic mouse model. Dystrophin expression remained stable and muscle-specific at all ages. The dystrophic phenotype was greatly ameliorated and, most importantly, muscle function in limb muscles was significantly improved not only in young adult but also in aged mice compared to nontransgenic littermates. Dystrophin expression was strong in fast-twitch skeletal muscles such as tibialis anterior and extensor digitorum longus, but weak or absent in heart, diaphragm, and slow-twitch muscles. Additionally, expression was strong in glycolytic but weak in oxidative fibers of fast-twitch muscles. This study may have important implications for the design of future gene therapy trials for muscular dystrophy.