Disease-associated polyalanine expansion mutations impair UBA6-dependent ubiquitination.

Expansion mutations in polyalanine stretches are associated with a growing number of diseases sharing a high degree of genotypic and phenotypic commonality. These similarities prompted us to query the normal function of physiological polyalanine stretches and to investigate whether a common molecular mechanism is involved in these diseases. Here, we show that UBA6, an E1 ubiquitin-activating enzyme, recognizes a polyalanine stretch within its cognate E2 ubiquitin-conjugating enzyme USE1. Aberrations in this polyalanine stretch reduce ubiquitin transfer to USE1 and, subsequently, polyubiquitination and degradation of its target, the ubiquitin ligase E6AP. Furthermore, we identify competition for the UBA6-USE1 interaction by various proteins with polyalanine expansion mutations in the disease state. The deleterious interactions of expanded polyalanine tract proteins with UBA6 in mouse primary neurons alter the levels and ubiquitination-dependent degradation of E6AP, which in turn affects the levels of the synaptic protein Arc. These effects are also observed in induced pluripotent stem cell-derived autonomic neurons from patients with polyalanine expansion mutations, where UBA6 overexpression increases neuronal resilience to cell death. Our results suggest a shared mechanism for such mutations that may contribute to the congenital malformations seen in polyalanine tract diseases.

Oculopharyngeal muscular dystrophy mutations link the RNA-binding protein HNRNPQ to autophagosome biogenesis.

Autophagy is an intracellular degradative process with an important role in cellular homeostasis. Here, we show that the RNA binding protein (RBP), heterogeneous nuclear ribonucleoprotein Q (HNRNPQ)/SYNCRIP is required to stimulate early events in autophagosome biogenesis, in particular the induction of VPS34 kinase by ULK1-mediated beclin 1 phosphorylation. The RBPs HNRNPQ and poly(A) binding protein nuclear 1 (PABPN1) form a regulatory network that controls the turnover of distinct autophagy-related (ATG) proteins. We also show that oculopharyngeal muscular dystrophy (OPMD) mutations engender a switch from autophagosome stimulation to autophagosome inhibition by impairing PABPN1 and HNRNPQ control of the level of ULK1. The overexpression of HNRNPQ in OPMD patient-derived cells rescues the defective autophagy in these cells. Our data reveal a regulatory mechanism of autophagy induction that is compromised by PABPN1 disease mutations, and may thus further contribute to their deleterious effects.

Transillumination Assisted Cricopharyngeal Myotomy.

Abstract: Cricopharyngeal muscle myotomy (CPM) is a common intervention for relief of dysphagia in patients with Oculo-pharyngeal muscular dystrophy (OPMD). Because of difficulties in approaching and dissecting cricopharyngeal muscle in these patients, we used transillumination for the myotomy (TA-CPM). Transillumination is a simple technique to improve the guidance and navigation of the surgeon in determining the location and depth of myotomy. The purpose of this study is to evaluate the efficacy and safety of transillumination in CPM in OPMD patients. An observational cohort of patients with OPMD who underwent CPM due to dysphagia at one medical center between 2010 and 2019. Two groups of patients are included, according to whether transillumination was used during their surgery. Patients were evaluated before and after surgery (1 week and 1 month) for their dysphagia score with a standardized questionnaire. The surgical team preferences, experience and complexity with and without transillumination were evaluated. Ten OPMD patients (8 heterozygotes, 2 homozygotes for the commonmutation) underwent CPM for relieving dysphagia symptoms at medium size medical center in Israel between 2010 and 2019. Five patients had TA-CPM and the 5 patients had CPM without transillumination. All patients showed an improvement at follow-up examinations, 1 week and 1 month postoperative, including a decrease in dysphagia score and in choking and aspiration events, compared to their preoperative state. TA-CPM improved the surgical approach, reduced the difficulty of CPM and was preferred by the surgical team. From the patients' point of view, TA-CPM was as good as a non-transillumination approach in improving dysphagia. TA-CPM is a cheap, fast and simple technique to improve the surgical outcomes in CPM for patients with OPMD. TA-CPM navigates the surgeon, helps with anatomical orientation, improve the surgeon's comfortable, may shorten the duration of surgery and reduces potential errors. Improvement in dysphagia score was similar in both groups. This technique may improve myotomy procedures for dysphagia of other etiologies. Level of evidence: IV. Case series (with or without comparison). Endoscopic transillumination assisted myotomy.

Assessment of PABPN1 nuclear inclusions on a large cohort of patients and in a human xenograft model of oculopharyngeal muscular dystrophy.

Oculopharyngeal muscular dystrophy (OPMD) is a rare muscle disease characterized by an onset of weakness in the pharyngeal and eyelid muscles. The disease is caused by the extension of a polyalanine tract in the Poly(A) Binding Protein Nuclear 1 (PABPN1) protein leading to the formation of intranuclear inclusions or aggregates in the muscle of OPMD patients. Despite numerous studies stressing the deleterious role of nuclear inclusions in cellular and animal OPMD models, their exact contribution to human disease is still unclear. In this study, we used a large and unique collection of human muscle biopsy samples to perform an in-depth analysis of PABPN1 aggregates in relation to age, genotype and muscle status with the final aim to improve our understanding of OPMD physiopathology. Here we demonstrate that age and genotype influence PABPN1 aggregates: the percentage of myonuclei containing PABPN1 aggregates increases with age and the chaperone HSP70 co-localize more frequently with PABPN1 aggregates with a larger polyalanine tract. In addition to the previously described PRMT1 and HSP70 co-factors, we identified new components of PABPN1 aggregates including GRP78/BiP, RPL24 and p62. We also observed that myonuclei containing aggregates are larger than myonuclei without. When comparing two muscles from the same patient, a similar amount of aggregates is observed in different muscles, except for the pharyngeal muscle where fewer aggregates are observed. This could be due to the peculiar nature of this muscle which has a low level of PAPBN1 and contains regenerating fibers. To confirm the fate of PABPN1 aggregates in a regenerating muscle, we generated a xenograft model by transplanting human OPMD muscle biopsy samples into the hindlimb of an immunodeficient mouse. Xenografts from subjects with OPMD displayed regeneration of human myofibers and PABPN1 aggregates were rapidly present-although to a lower extent-after muscle fiber regeneration. Our data obtained on human OPMD samples add support to the dual non-exclusive models in OPMD combining toxic PABPN1 intranuclear inclusions together with PABPN1 loss of function which altogether result in this late-onset and muscle selective disease.

Oculopharyngeal Muscular Dystrophy and Inherited Retinal Dystrophy in Bukhara Jews Due to Linked Mutations in the PABPN1 and NRL Genes.

AIM: We have previously described two unrelated Bukhara Jews (BJs) with a combination of oculopharyngeal muscular dystrophy (OPMD) and inherited retinal dystrophy (IRD), because of mutations in two linked genes: PABPN1 and NRL. Here we investigated the prevalence of the NRL mutation among BJs with OPMD. MATERIALS AND METHODS: PABPN1 and NRL mutation testing were performed by polymerase chain reaction amplification and direct sequencing on two cohorts of Bukhara Jewish patients: OPMD patients (with or without IRD) and IRD patients (without OPMD). RESULTS: Of 24 unrelated chromosomes from Bukhara Jewish OPMD patients, 19 (79%) harbored the NRL mutation. In contrast, the NRL mutation was not detected in Bukhara Jewish patients diagnosed with IRD but without OPMD. CONCLUSIONS: Our findings provide an explanation for the reoccurrence of IRD in Bukhara Jewish OPMD homozygotes. Moreover, they indicate that Bukhara Jewish OPMD patients are at high risk for carrying the NRL mutation, and should be offered appropriate genetic counseling and testing.

Large Intragenic Deletion in DSTYK Underlies Autosomal-Recessive Complicated Spastic Paraparesis, SPG23.

SPG23 is an autosomal-recessive neurodegenerative subtype of lower limb spastic paraparesis with additional diffuse skin and hair dyspigmentation at birth followed by further patchy pigment loss during childhood. Previously, genome-wide linkage in an Arab-Israeli pedigree mapped the gene to an approximately 25 cM locus on chromosome 1q24-q32. By using whole-exome sequencing in a further Palestinian-Jordanian SPG23 pedigree, we identified a complex homozygous 4-kb deletion/20-bp insertion in DSTYK (dual serine-threonine and tyrosine protein kinase) in all four affected family members. DSTYK is located within the established linkage region and we also found the same mutation in the previously reported pedigree and another Israeli pedigree (total of ten affected individuals from three different families). The mutation removes the last two exons and part of the 3' UTR of DSTYK. Skin biopsies revealed reduced DSTYK protein levels along with focal loss of melanocytes. Ultrastructurally, swollen mitochondria and cytoplasmic vacuoles were also noted in remaining melanocytes and some keratinocytes and fibroblasts. Cultured keratinocytes and fibroblasts from an affected individual, as well as knockdown of Dstyk in mouse melanocytes, keratinocytes, and fibroblasts, were associated with increased cell death after ultraviolet irradiation. Keratinocytes from an affected individual showed loss of kinase activity upon stimulation with fibroblast growth factor. Previously, dominant mutations in DSTYK were implicated in congenital urological developmental disorders, but our study identifies different phenotypic consequences for a recurrent autosomal-recessive deletion mutation in revealing the genetic basis of SPG23.

Homozygosity for a Recessive Loss-of-Function Mutation of the NRL Gene Is Associated With a Variant of Enhanced S-Cone Syndrome.

PURPOSE: To investigate the genetic basis for severe visual complaints by Bukharan Jewish patients with oculopharyngeal muscular dystrophy (OPMD). METHODS: Polymerase chain reaction amplification and direct sequencing were used to test for NRL, PABPN1, and NR2E3 mutations. Complete ophthalmic examination included best-corrected visual acuity, biomicroscopic examination, optical coherence tomography, and fundus autofluorescence. Detailed electroretinography (ERG) testing was conducted including expanded International Society for Clinical Electrophysiology of Vision protocol for light-adapted and dark-adapted conditions, measurements of S-cone function, and ON-OFF light-adapted ERG. RESULTS: The index patients were homozygotes for both a dominant mutation of the PABPN1 gene, (GCN)13, and a recessive mutation of the NRL gene, p.R31X, on chromosome 14q11.1, leading to early-onset OPMD accompanied by night blindness and reduced visual acuity. No mutations were found in the NR2E3 gene. Both patients were of Bukharan Jewish origin, but from unrelated families. Electroretinography responses of both patients were dominated by short-wavelength-sensitive mechanisms, with no detectable rod function, similar to the ERG responses of individuals with enhanced S-cone syndrome (ESCS) due to NR2E3 mutations. Heterozygotes for the PABPN1 and NRL mutations demonstrated normal fundi and ERG responses. CONCLUSIONS: Homozygosity for the recessive NRL mutation described here appears to be associated with a distinct retinal phenotype, demonstrating ERG characteristics similar to those of ESCS patients. This report expands the spectrum of NRL recessive mutations, as well as the genetic spectrum of ESCS, and indicates a new syndrome of OPMD with an ESCS-like phenotype.

Dynamic MRI testing of the cervical spine has prognostic significance in patients with progressive upper-limb distal weakness and atrophy.

BACKGROUND: The syndrome of isolated progressive upper-limb distal weakness and atrophy results from disease processes affecting lower motor neurons originating in the cervical anterior horn gray matter. Lower motor neuron dysfunction restricted to the C7-T1 myotomes in the absence of neuropathy, upper motor neuron signs, sphincter disturbances or abnormality on conventional MR imaging is suggestive of either Hirayama disease (HD), or the initial manifestation of a progressive motor neuron disease such as amyotrophic lateral sclerosis (ALS). In HD the supposed etiologic mechanism is a mechanical compression of the cervical spinal cord during neck flexion; therefore, dynamic MRI (dMRI) of the cervical cord might help differentiate between these possibilities. METHODS: This was a multi-center observational cohort study. Over a 4-year period between 8/2009 and 8/2013, 22 patients were identified as having a disease consistent with HD. We identified a subgroup of patients suspected of suffering from active progressive disease and prospectively followed them after performing dynamic MRI studies of the cervical spine. RESULTS: Twenty-two patients were identified as having a disease consistent with HD, of whom 8 were defined as having actively progressive disease. Seven of these 8 patients demonstrated clear dynamic compression of the cervical spine during neck flexion. The patient who did not demonstrate the typical MRI changes associated with HD went on to develop generalized ALS. CONCLUSIONS: dMRI has a practical role in patients presenting with progressive upper-limb distal weakness and atrophy, and the presence of characteristic changes typical of HD may suggest a more optimistic prognosis.

Oculopharyngeal muscular dystrophy among Bulgarian Jews: a new cluster?

BACKGROUND: Oculopharyngeal muscular dystrophy (OPMD) produced by the (GCG)13 expansion mutation in the PABPN1 gene is frequent among Uzbek Jews in Israel. OBJECTIVES: To describe the phenotypic and genotypic features in five Bulgarian Jewish patients, from different families, with autosomal dominant OPMD. METHODS: We performed clinical follow-up, electrodiagnostic tests and mutation detection. Blood samples were obtained after informed consent and DNA was extracted; measurement of GCG repeats in both PABPN1 alleles and sequencing of OPMD mutations were performed according to standard techniques. RESULTS: We identified five patients (four females), aged 58 to 71 years, with bilateral ptosis, dysphagia, dysphonia (n = 3) and myopathic motor units by electromyography. In all patients we noticed proximal weakness of the upper limbs with winging scapulae in three of them. All cases shared the (GCG)13-(GCG)10 PABPN1 genotype. CONCLUSIONS: OPMD among Bulgarian Jews is produced by a (GCG)13 expansion, identical to the mutation in Uzbek Jews and French Canadians. In addition to the classical neurological and neuro-ophthalmological features, early shoulder girdle weakness is common in Bulgarian Jewish patients; this is an unusual feature during the early stages of OPMD produced by the same mutation in other populations. We suggest that besides the disease-producing GCG expansion, additional ethnicity-related genetic factors may influence the OPMD phenotype. OPMD is a rare disease, and the identification of five affected families in the rather small Bulgarian Jewish community in Israel probably represents a new cluster; future haplotype studies may elucidate whether a founder effect occurred.

Novel mutation in VCP gene causes atypical amyotrophic lateral sclerosis.

OBJECTIVE: To identify the genetic variant that causes autosomal dominantly inherited motor neuron disease in a 4-generation Israeli-Arab family using genetic linkage and whole exome sequencing. METHODS: Genetic linkage analysis was performed in this family using Illumina single nucleotide polymorphism chips. Whole exome sequencing was then undertaken on DNA samples from 2 affected family members using an Illumina 2000 HiSeq platform in pursuit of potentially pathogenic genetic variants that comigrate with the disease in this pedigree. Variants meeting these criteria were then screened in all affected individuals. RESULTS: A novel mutation (p.R191G) in the valosin-containing protein (VCP) gene was identified in the index family. Direct sequencing of the VCP gene in a panel of DNA from 274 unrelated individuals with familial amyotrophic lateral sclerosis (FALS) revealed 5 additional mutations. Among them, 2 were previously identified in pedigrees with a constellation of inclusion body myopathy with Paget disease of the bone and frontotemporal dementia (IBMPFD) and in FALS, and 2 other mutations (p.R159C and p.R155C) in IBMPFD alone. We did not detect VCP gene mutations in DNA from 178 cases of sporadic amyotrophic lateral sclerosis. CONCLUSIONS: We report a novel VCP mutation identified in an amyotrophic lateral sclerosis family (p.R191G) with atypical clinical features. In our experience, VCP mutations arise in approximately 1.5% of FALS cases. Our study supports the view that motor neuron disease is part of the clinical spectrum of VCP-associated disease.

A rare recessive distal hereditary motor neuropathy with HSJ1 chaperone mutation.

OBJECTIVE: Distal hereditary motor neuropathies (dHMN) form a clinically and genetically heterogeneous group of disorders, characterized by muscle weakness and atrophy predominating at the distal part of the limbs, due to the progressive degeneration of motor neurons in the spinal cord. We report here a novel rare variant of dHMN with autosomal recessive inheritance in a large Jewish family originating from Morocco. The disease is characterized by a predominance of paralysis at the lower limbs and an early adulthood onset. We performed a genetic study in this family to identify and characterized the causing mutation. METHODS: Homozygosity mapping strategy and sequencing of the candidate genes were performed. Expression studies were made on patient fibroblasts. Functional experiments were performed on a cellular model of motor neuron disease. RESULTS: We mapped the disease to the 2q34-q36.1 chromosomal region and identified a homozygous splice mutation in the gene HSJ1 (DNAJB2) decreasing the expression of the 2 main isoforms HSJ1a and HSJ1b. Overexpression of both HSJ1a and HSJ1b reduced inclusion formation induced by the mutated SOD1-A4V in a neuronal cellular model. INTERPRETATION: HSJ1 is a neuronal enriched member of the HSP40/DNAJ co-chaperone family. Previous studies have shown that HSP40 proteins play a crucial role in protein aggregation and neurodegeneration in several neuronal types, in animal models and human diseases. Interestingly, this mutation causing a loss-of-function of HSJ1 is linked to a pure lower motor neuron disease, strongly suggesting that HSJ1 also plays an important and specific role in motor neurons.

Immortalized pathological human myoblasts: towards a universal tool for the study of neuromuscular disorders.

BACKGROUND: Investigations into both the pathophysiology and therapeutic targets in muscle dystrophies have been hampered by the limited proliferative capacity of human myoblasts. Isolation of reliable and stable immortalized cell lines from patient biopsies is a powerful tool for investigating pathological mechanisms, including those associated with muscle aging, and for developing innovative gene-based, cell-based or pharmacological biotherapies. METHODS: Using transduction with both telomerase-expressing and cyclin-dependent kinase 4-expressing vectors, we were able to generate a battery of immortalized human muscle stem-cell lines from patients with various neuromuscular disorders. RESULTS: The immortalized human cell lines from patients with Duchenne muscular dystrophy, facioscapulohumeral muscular dystrophy, oculopharyngeal muscular dystrophy, congenital muscular dystrophy, and limb-girdle muscular dystrophy type 2B had greatly increased proliferative capacity, and maintained their potential to differentiate both in vitro and in vivo after transplantation into regenerating muscle of immunodeficient mice. CONCLUSIONS: Dystrophic cellular models are required as a supplement to animal models to assess cellular mechanisms, such as signaling defects, or to perform high-throughput screening for therapeutic molecules. These investigations have been conducted for many years on cells derived from animals, and would greatly benefit from having human cell models with prolonged proliferative capacity. Furthermore, the possibility to assess in vivo the regenerative capacity of these cells extends their potential use. The innovative cellular tools derived from several different neuromuscular diseases as described in this report will allow investigation of the pathophysiology of these disorders and assessment of new therapeutic strategies.

Mutational analysis of glycyl-tRNA synthetase (GARS) gene in Hirayama disease.

Sporadic juvenile muscular atrophy of the distal upper extremity or Hirayama's disease (HD) and autosomal dominant motor distal neuronopathy/axonopathy (CMT2D/dSMA-V), produced by glycyl-tRNA synthetase (GARS) gene mutations, share some clinical features including: young age of onset, predilection for the distal upper extremity, asymmetry, sparing of proximal muscles and unusual cold sensitivity. However, incomplete penetrance of GARS gene mutations may account for apparently non-familial cases. In order to inquire whether GARS gene mutations are associated with HD we studied seven patients fulfilling the clinical and electrodiagnostic criteria for HD. All patients underwent MRI of cervical spine that excluded compressive myelopathy in neutral position and intramedullary pathology. Each patient was tested for the presence of mutations in GARS by sequencing all coding exons amplified from genomic DNA. No pathogenic mutations were found, excluding the role of GARS gene as a possible factor in the aetiology of HD in this cohort.

Aggressive familial ALS with unusual brain MRI and a SOD1 gene mutation.

We studied two sisters with rapidly progressing ALS starting at the ages of 46 and 48 years and leading to death after 14 months. Both fulfilled the El Escorial criteria for definite ALS and had marked upper motor neuron (UMN) predominance. Brain MRI, on fluid attenuation recovery (FLAIR) mode, showed outstanding hyperintensities of the precentral gyrus, centrum semiovale, corona radiata and along the corticospinal pathways in the brainstem. Screening for the SOD1 gene disclosed, at codon 140, a base substitution of adenine for thymine (GGT>CCA) known as the A140A 'silent' mutation since it does not change the amino acid (alanine) encoded for at that position. The severe UMN involvement and the fast progression of the disease may correlate with the MRI findings. It is also possible that the A140A mutation is not incidental; the mutated mRNA might be cytotoxic.

Apparent weakness of ulnar-innervated muscles in radial palsy.

Muscle strength was assessed in 11 patients with radial or posterior interosseus palsy. Apparent weakness was found in the dorsal and palmar interossei and the abductor digiti minimi. These muscles insert on the extensor expansions, and their activation is associated with concomitant contraction of finger flexors and extensors. This apparent weakness may be due to their unopposed traction on the extensor expansion by the paralyzed extensor digitorum.

A locus for complicated hereditary spastic paraplegia maps to chromosome 1q24-q32.

We updated the clinical features of a consanguineous Arab Israeli family, in which four of seven children were affected by spastic paraplegia complicated by skin pigmentary abnormalities. A genomewide linkage screen performed for the family identified a new locus (SPG23) for this form of hereditary spastic paraplegia, in an approximately 25cM region of chromosome 1q24-q32, with a peak logarithm of odds score of 3.05.

From the philosophy auditorium to the neurophysiology laboratory and back: from Bergson to Damasio.

Henri Bergson (1859-1941) was probably the most influential French philosopher at the turn of the twentieth century. In 1927 he was awarded the Nobel Prize for literature. Far beyond the restricted academic philosophical milieu, the impact of his thinking reached personalities as diverse as Claude Debussy, Marcel Proust, George Bemard Shaw, and the impressionists. His essay The Laughter (Le Rire) is one of the most profound and original ever written on the sense of humor. Bergson's opinions, with their emphasis on life, instinct and intuition, represented a deviation from the rationalist mainstream of western philosophical tradition. In some circles he was received with skepticism and irony, as in Bertrand Russel's History of Western Philosophy. Today, unbiased by theoretical "bergsonism," neurophysiologic research--as undertaken mainly by Antonio Damasio's team at Iowa University--confirms many of his hypotheses and elucidates their mechanisms. In this new light, intuition and "recognition by the body" should not be seen as the personal fantasy of an original thinker but as fundamental cognitive tools.