ABSTRACT
Spinocerebellar ataxia 27B (SCA27B) is a common autosomal dominant ataxia caused by an intronic GAAâ¢TTC repeat expansion in FGF14 . Neuropathological studies have shown that neuronal loss is largely restricted to the cerebellum. Although the repeat locus is highly unstable during intergenerational transmission, it remains unknown whether it exhibits cerebral mosaicism and progressive instability throughout life. We conducted an analysis of the FGF14 GAAâ¢TTC repeat somatic instability across 156 serial blood samples from 69 individuals, fibroblasts, induced pluripotent stem cells, and post-mortem brain tissues from six controls and six patients with SCA27B, alongside methylation profiling using targeted long-read sequencing. Peripheral tissues exhibited minimal somatic instability, which did not significantly change over periods of more than 20 years. In post-mortem brains, the GAAâ¢TTC repeat was remarkably stable across all regions, except in the cerebellar hemispheres and vermis. The levels of somatic expansion in the cerebellar hemispheres and vermis were, on average, 3.15 and 2.72 times greater relative to other examined brain regions, respectively. Additionally, levels of somatic expansion in the brain increased with repeat length and tissue expression of FGF14 . We found no significant difference in methylation of wild-type and expanded FGF14 alleles in post-mortem cerebellar hemispheres between patients and controls. In conclusion, our study revealed that the FGF14 GAAâ¢TTC repeat exhibits a cerebellar-specific expansion bias, which may explain the pure and late-onset cerebellar involvement in SCA27B.
ABSTRACT
BACKGROUND: SCA27B caused by FGF14 intronic heterozygous GAA expansions with at least 250 repeats accounts for 10-60% of cases with unresolved cerebellar ataxia. We aimed to assess the size and frequency of FGF14 expanded alleles in individuals with cerebellar ataxia as compared with controls and to characterize genetic and clinical variability. METHODS: We sized this repeat in 1876 individuals from France sampled for research purposes in this cross-sectional study: 845 index cases with cerebellar ataxia and 324 affected relatives, 475 controls, as well as 119 cases with spastic paraplegia, and 113 with familial essential tremor. FINDINGS: A higher frequency of expanded allele carriers in index cases with ataxia was significant only above 300 GAA repeats (10.1%, n = 85) compared with controls (1.1%, n = 5) (p < 0.0001) whereas GAA250-299 alleles were detected in 1.7% of both groups. Eight of 14 index cases with GAA250-299 repeats had other causal pathogenic variants (4/14) and/or discordance of co-segregation (5/14), arguing against GAA causality. We compared the clinical signs in 127 GAA≥300 carriers to cases with non-expanded GAA ataxia resulting in defining a key phenotype triad: onset after 45 years, downbeat nystagmus, episodic ataxic features including diplopia; and a frequent absence of dysarthria. All maternally transmitted alleles above 100 GAA were unstable with a median expansion of +18 repeats per generation (r2 = 0.44; p < 0.0001). In comparison, paternally transmitted alleles above 100 GAA mostly decreased in size (-15 GAA (r2 = 0.63; p < 0.0001)), resulting in the transmission bias observed in SCA27B pedigrees. INTERPRETATION: SCA27B diagnosis must consider both the phenotype and GAA expansion size. In carriers of GAA250-299 repeats, the absence of documented familial transmission and a presentation deviating from the key SCA27B phenotype, should prompt the search for an alternative cause. Affected fathers have a reduced risk of having affected children, which has potential implications for genetic counseling. FUNDING: This work was supported by the Fondation pour la Recherche Médicale, grant number 13338 to JLM, the Association Connaître les Syndrome Cérébelleux - France (to GS) and by the European Union's Horizon 2020 research and innovation program under grant agreement No 779257 ("SOLVE-RD" to GS). DP holds a Fellowship award from the Canadian Institutes of Health Research (CIHR). SK received a grant (01GM1905C) from the Federal Ministry of Education and Research, Germany, through the TreatHSP network. This work was supported by the Australian Government National Health and Medical Research Council grants (GNT2001513 and MRFF2007677) to MB and PJL.
Subject(s)
Cerebellar Ataxia , Friedreich Ataxia , Child , Humans , Ataxia/diagnosis , Ataxia/genetics , Australia , Canada , Cerebellar Ataxia/diagnosis , Cerebellar Ataxia/genetics , Cross-Sectional Studies , Friedreich Ataxia/geneticsABSTRACT
Usually, molecular diagnosis of spinocerebellar ataxia is based on a step-by-step approach with targeted sizing of four repeat expansions accounting for most dominant cases, then targeted sequencing of other genes. Nowadays, genome sequencing allows detection of most pathogenic variants in a single step. The ExpansionHunter tool can detect expansions in short-read genome sequencing data. Recent studies have shown that ExpansionHunter can also be used to identify repeat expansions in exome sequencing data. We tested ExpansionHunter on spinocerebellar ataxia exomes in a research context as a second-line analysis, after exclusion of main CAG repeat expansions in half of the probands. First, we confirmed the detection of expansions in seven known expansion carriers and then, after targeted analysis of ATXN1, 2, 3 and 7, CACNA1A, TBP, ATN1, NOP56, AR and HTT in 498 exomes, we found 22 additional pathogenic expansions. Comparison with capillary migration sizing in 247 individuals and confirmation of all expanded alleles detected by ExpansionHunter demonstrated that for these loci, sensitivity and specificity reached 100%. ExpansionHunter detected but underestimated the repeat size for larger expansions, and the normal alleles distribution at each locus should be taken into account to detect expansions. Exome combined with ExpansionHunter is reliable to detect repeat expansions in selected loci as first-line analysis in spinocerebellar ataxia.
Subject(s)
Exome , Spinocerebellar Ataxias , Humans , Exome/genetics , Spinocerebellar Ataxias/diagnosis , Spinocerebellar Ataxias/genetics , Alleles , HeterozygoteABSTRACT
PURPOSE: CAG/CAA repeat expansions in TBP>49 are responsible for spinocerebellar ataxia (SCA) type 17 (SCA17). We previously detected cosegregation of STUB1 variants causing SCA48 with intermediate alleles of TBP in 2 families. This cosegregation questions the existence of SCA48 as a monogenic disease. METHODS: We systematically sequenced TBP repeats in 34 probands of dominant ataxia families with STUB1 variants. In addition, we searched for pathogenic STUB1 variants in probands with expanded alleles of TBP>49 (n = 2) or intermediate alleles of TBP≥40 (n = 47). RESULTS: STUB1 variants were found in half of the TBP40-49 cohort. Mirroring this finding, TBP40-49 alleles were detected in 40% of STUB1 probands. The longer the TBP repeat length, the more likely the occurrence of cognitive impairment (P = .0129) and the faster the disease progression until death (P = .0003). Importantly, 13 STUB1 probands presenting with the full SCA48 clinical phenotype had normal TBP37-39 alleles, excluding digenic inheritance as the sole mode. CONCLUSION: We show that intermediate TBP40-49 alleles act as disease modifiers of SCA48 rather than a STUB1/TBP digenic model. This distinction from what has been proposed before has crucial consequences for genetic counseling in SCA48.
Subject(s)
Cerebellar Ataxia , Spinocerebellar Ataxias , Humans , Spinocerebellar Ataxias/genetics , Spinocerebellar Ataxias/pathology , Cerebellar Ataxia/genetics , Phenotype , Alleles , Trinucleotide Repeat Expansion/genetics , Ubiquitin-Protein Ligases/geneticsABSTRACT
Background and Objectives: Neurodevelopmental and neurodegenerative disorders have long been considered as different clinical and molecular entities, and only a few genes are known to be involved in both processes. The IRF2BPL (interferon regulatory factor 2 binding protein like) gene was implicated in a severe pediatric phenotype characterized by developmental and epileptic encephalopathy and early regression. In parallel, inherited IRF2BPL variants have been reported in cohorts of patients with late-onset progressive dystonic and ataxic syndrome with few information about the neurodevelopment of these patients. This study aimed to describe both neurodevelopmental and neurodegenerative aspects of the phenotype in adults with IRF2BPL pathogenic variant. Methods: We report here the clinical and molecular data of 18 individuals carrying truncating IRF2BPL variants (identified by either exome or genome sequencing), including a large pedigree of 16 patients presenting with a neurodevelopmental disorder (NDD) associated with late-onset cerebellar ataxia and atrophy. Results: Genome sequencing identified the p.(Gln117*) variant in a large family first assessed for familial ataxia, with multiple individuals presenting with NDD. The p.(Ser313*) variant was identified by exome sequencing in a second family with a young adult patient with NDD without ataxia which was inherited from her asymptomatic mother, suggesting incomplete penetrance of IRF2BPL-linked disorders. Discussion: This study illustrates the importance of neurologic evaluation of adult patients initially diagnosed with NDD to detect a late-onset neurodegenerative condition. Two different disorders may be clinically diagnosed in the same family, when not considering that NDD and late cerebellar changes may be part of the same molecular spectrum such as for IRF2BPL.
ABSTRACT
PURPOSE: Pathogenic variants in STUB1 were initially described in autosomal recessive spinocerebellar ataxia type 16 and dominant cerebellar ataxia with cerebellar cognitive dysfunction (SCA48). METHODS: We analyzed a large series of 440 index cerebellar ataxia cases, mostly with dominant inheritance. RESULTS: STUB1 variants were detected in 50 patients. Age at onset and severity were remarkably variable. Cognitive impairment, predominantly frontal syndrome, was observed in 54% of STUB1 variant carriers, including five families with Huntington or frontotemporal dementia disease-like phenotypes associated with ataxia, while no STUB1 variant was found in 115 patients with frontotemporal dementia. We report neuropathological findings of a STUB1 heterozygous patient, showing massive loss of Purkinje cells in the vermis and major loss in the cerebellar hemispheres without atrophy of the pons, hippocampus, or cerebral cortex. This screening of STUB1 variants revealed new features: (1) the majority of patients were women (70%) and (2) "second hits" in AFG3L2, PRKCG, and TBP were detected in three families suggesting synergic effects. CONCLUSION: Our results reveal an unexpectedly frequent (7%) implication of STUB1 among dominantly inherited cerebellar ataxias, and suggest that the penetrance of STUB1 variants could be modulated by other factors, including sex and variants in other ataxia-related genes.
Subject(s)
Cerebellar Ataxia , Cognitive Dysfunction , Spinocerebellar Ataxias , ATP-Dependent Proteases , ATPases Associated with Diverse Cellular Activities , Ataxia , Cerebellar Ataxia/genetics , Female , Humans , Male , Spinocerebellar Ataxias/genetics , Ubiquitin-Protein LigasesABSTRACT
OBJECTIVE: We took advantage of a large multinational recruitment to delineate genotype-phenotype correlations in a large, trans-European multicenter cohort of patients with spastic paraplegia gene 7 (SPG7). METHODS: We analyzed clinical and genetic data from 241 patients with SPG7, integrating neurologic follow-up data. One case was examined neuropathologically. RESULTS: Patients with SPG7 had a mean age of 35.5 ± 14.3 years (n = 233) at onset and presented with spasticity (n = 89), ataxia (n = 74), or both (n = 45). At the first visit, patients with a longer disease duration (>20 years, n = 62) showed more cerebellar dysarthria (p < 0.05), deep sensory loss (p < 0.01), muscle wasting (p < 0.01), ophthalmoplegia (p < 0.05), and sphincter dysfunction (p < 0.05) than those with a shorter duration (<10 years, n = 93). Progression, measured by Scale for the Assessment and Rating of Ataxia evaluations, showed a mean annual increase of 1.0 ± 1.4 points in a subgroup of 30 patients. Patients homozygous for loss of function (LOF) variants (n = 65) presented significantly more often with pyramidal signs (p < 0.05), diminished visual acuity due to optic atrophy (p < 0.0001), and deep sensory loss (p < 0.0001) than those with at least 1 missense variant (n = 176). Patients with at least 1 Ala510Val variant (58%) were older (age 37.6 ± 13.7 vs 32.8 ± 14.6 years, p < 0.05) and showed ataxia at onset (p < 0.05). Neuropathologic examination revealed reduction of the pyramidal tract in the medulla oblongata and moderate loss of Purkinje cells and substantia nigra neurons. CONCLUSIONS: This is the largest SPG7 cohort study to date and shows a spasticity-predominant phenotype of LOF variants and more frequent cerebellar ataxia and later onset in patients carrying at least 1 Ala510Val variant.
Subject(s)
ATPases Associated with Diverse Cellular Activities/genetics , Cerebellar Ataxia/genetics , Metalloendopeptidases/genetics , Paraplegia/genetics , Spastic Paraplegia, Hereditary/genetics , Adult , Cerebellar Ataxia/physiopathology , Cohort Studies , Electromyography , Female , Humans , Loss of Function Mutation , Magnetic Resonance Imaging , Male , Middle Aged , Paraplegia/physiopathology , Phenotype , Polymorphism, Single Nucleotide , Spastic Paraplegia, Hereditary/physiopathology , White People/genetics , Young AdultABSTRACT
Importance: Molecular diagnosis is difficult to achieve in disease groups with a highly heterogeneous genetic background, such as cerebellar ataxia (CA). In many patients, candidate gene sequencing or focused resequencing arrays do not allow investigators to reach a genetic conclusion. Objectives: To assess the efficacy of exome-targeted capture sequencing to detect mutations in genes broadly linked to CA in a large cohort of undiagnosed patients and to investigate their prevalence. Design, Setting, and Participants: Three hundred nineteen index patients with CA and without a history of dominant transmission were included in the this cohort study by the Spastic Paraplegia and Ataxia Network. Centralized storage was in the DNA and cell bank of the Brain and Spine Institute, Salpetriere Hospital, Paris, France. Patients were classified into 6 clinical groups, with the largest being those with spastic ataxia (ie, CA with pyramidal signs [n = 100]). Sequencing was performed from January 1, 2014, through December 31, 2016. Detected variants were classified as very probably or definitely causative, possibly causative, or of unknown significance based on genetic evidence and genotype-phenotype considerations. Main Outcomes and Measures: Identification of variants in genes broadly linked to CA, classified in pathogenicity groups. Results: The 319 included patients had equal sex distribution (160 female [50.2%] and 159 male patients [49.8%]; mean [SD] age at onset, 27.9 [18.6] years). The age at onset was younger than 25 years for 131 of 298 patients (44.0%) with complete clinical information. Consanguinity was present in 101 of 298 (33.9%). Very probable or definite diagnoses were achieved for 72 patients (22.6%), with an additional 19 (6.0%) harboring possibly pathogenic variants. The most frequently mutated genes were SPG7 (n = 14), SACS (n = 8), SETX (n = 7), SYNE1 (n = 6), and CACNA1A (n = 6). The highest diagnostic rate was obtained for patients with an autosomal recessive CA with oculomotor apraxia-like phenotype (6 of 17 [35.3%]) or spastic ataxia (35 of 100 [35.0%]) and patients with onset before 25 years of age (41 of 131 [31.3%]). Peculiar phenotypes were reported for patients carrying KCND3 or ERCC5 variants. Conclusions and Relevance: Exome capture followed by targeted analysis allows the molecular diagnosis in patients with highly heterogeneous mendelian disorders, such as CA, without prior assumption of the inheritance mode or causative gene. Being commonly available without specific design need, this procedure allows testing of a broader range of genes, consequently describing less classic phenotype-genotype correlations, and post hoc reanalysis of data as new genes are implicated in the disease.
Subject(s)
Cerebellar Ataxia/genetics , Exome Sequencing/methods , Genetic Predisposition to Disease , Mutation/genetics , ATPases Associated with Diverse Cellular Activities/genetics , Adolescent , Adult , Calcium Channels/genetics , Cohort Studies , Computational Biology , Cytoskeletal Proteins , DNA Helicases , Female , Heat-Shock Proteins/genetics , Humans , Male , Metalloendopeptidases/genetics , Middle Aged , Multifunctional Enzymes , Nerve Tissue Proteins/genetics , Nuclear Proteins/genetics , Phenotype , RNA Helicases/genetics , Young AdultABSTRACT
Mutations in GFPT1 (glutamine-fructose-6-phosphate transaminase 1), a gene encoding an enzyme involved in glycosylation of ubiquitous proteins, cause a limb-girdle congenital myasthenic syndrome (LG-CMS) with tubular aggregates (TAs) characterized predominantly by affection of the proximal skeletal muscles and presence of highly organized and remodeled sarcoplasmic tubules in patients' muscle biopsies. We report here the first long-term clinical follow-up of 11 French individuals suffering from LG-CMS with TAs due to GFPT1 mutations, of which nine are new. Our retrospective clinical evaluation stresses an evolution toward a myopathic weakness that occurs concomitantly to ineffectiveness of usual CMS treatments. Analysis of neuromuscular biopsies from three unrelated individuals demonstrates that the maintenance of neuromuscular junctions (NMJs) is dramatically impaired with loss of post-synaptic junctional folds and evidence of denervation-reinnervation processes affecting the three main NMJ components. Moreover, molecular analyses of the human muscle biopsies confirm glycosylation defects of proteins with reduced O-glycosylation and show reduced sialylation of transmembrane proteins in extra-junctional area. Altogether, these results pave the way for understanding the etiology of this rare neuromuscular disorder that may be considered as a "tubular aggregates myopathy with synaptopathy".
Subject(s)
Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing)/genetics , Myasthenic Syndromes, Congenital/genetics , Myasthenic Syndromes, Congenital/pathology , Myopathies, Structural, Congenital/genetics , Myopathies, Structural, Congenital/pathology , Neuromuscular Junction/pathology , Adolescent , Adult , Aged , Female , Follow-Up Studies , Glycosylation , Humans , Middle Aged , Muscle, Skeletal/enzymology , Muscle, Skeletal/innervation , Muscle, Skeletal/pathology , Myasthenic Syndromes, Congenital/drug therapy , Myasthenic Syndromes, Congenital/enzymology , Myopathies, Structural, Congenital/drug therapy , Myopathies, Structural, Congenital/enzymology , Neuromuscular Junction/enzymology , Prospective Studies , Retrospective Studies , Young AdultABSTRACT
Autosomal dominant cerebellar ataxias have a marked heterogeneous genetic background, with mutations in 34 genes identified so far. This large amount of implicated genes accounts for heterogeneous clinical presentations, making genotype-phenotype correlations a major challenge in the field. While polyglutamine ataxias, linked to CAG repeat expansions in genes such as ATXN1, ATXN2, ATXN3, ATXN7, CACNA1A and TBP, have been extensively characterized in large cohorts, there is a need for comprehensive assessment of frequency and phenotype of more 'conventional' ataxias. After exclusion of CAG/polyglutamine expansions in spinocerebellar ataxia genes in 412 index cases with dominantly inherited cerebellar ataxias, we aimed to establish the relative frequencies of mutations in other genes, with an approach combining panel sequencing and TaqMan® polymerase chain reaction assay. We found relevant genetic variants in 59 patients (14.3%). The most frequently mutated were channel genes [CACNA1A (n = 16), KCND3 (n = 4), KCNC3 (n = 2) and KCNA1 (n = 2)]. Deletions in ITPR1 (n = 11) were followed by biallelic variants in SPG7 (n = 9). Variants in AFG3L2 (n = 7) came next in frequency, and variants were rarely found in STBN2 (n = 2), ELOVL5, FGF14, STUB1 and TTBK2 (n = 1 each). Interestingly, possible risk factor variants were detected in SPG7 and POLG. Clinical comparisons showed that ataxias due to channelopathies had a significantly earlier age at onset with an average of 24.6 years, versus 40.9 years for polyglutamine expansion spinocerebellar ataxias and 37.8 years for SPG7-related forms (P = 0.001). In contrast, disease duration was significantly longer in the former (20.5 years versus 9.3 and 13.7, P=0.001), though for similar functional stages, indicating slower progression of the disease. Of interest, intellectual deficiency was more frequent in channel spinocerebellar ataxias, while cognitive impairment in adulthood was similar among the three groups. Similar differences were found among a single gene group, comparing 23 patients with CACNA1A expansions (spinocerebellar ataxia 6) to 22 patients with CACNA1A point mutations, which had lower average age at onset (25.2 versus 47.3 years) with longer disease duration (18.7 versus 10.9), but lower severity indexes (0.39 versus 0.44), indicating slower progression of the disease. In conclusion, we identified relevant genetic variations in up to 15% of cases after exclusion of polyglutamine expansion spinocerebellar ataxias, and confirmed CACNA1A and SPG7 as major ataxia genes. We could delineate firm genotype-phenotype correlations that are important for genetic counselling and of possible prognostic value.
Subject(s)
Calcium Channels/genetics , Cerebellar Ataxia/genetics , Cerebellar Ataxia/physiopathology , Channelopathies/genetics , Channelopathies/physiopathology , Metalloendopeptidases/genetics , ATPases Associated with Diverse Cellular Activities , Adolescent , Adult , Age of Onset , Aged , Aged, 80 and over , Child , Child, Preschool , Cohort Studies , Female , Genes, Dominant , Genotype , Humans , Male , Middle Aged , Phenotype , Young AdultABSTRACT
Schwartz-Jampel syndrome (SJS) is a recessive disorder with muscle hyperactivity that results from hypomorphic mutations in the perlecan gene, a basement membrane proteoglycan. Analyses done on a mouse model have suggested that SJS is a congenital form of distal peripheral nerve hyperexcitability resulting from synaptic acetylcholinesterase deficiency, nerve terminal instability with preterminal amyelination, and subtle peripheral nerve changes. We investigated one adult patient with SJS to study this statement in humans. Perlecan deficiency due to hypomorphic mutations was observed in the patient biological samples. Electroneuromyography showed normal nerve conduction, neuromuscular transmission, and compound nerve action potentials while multiple measures of peripheral nerve excitability along the nerve trunk did not detect changes. Needle electromyography detected complex repetitive discharges without any evidence for neuromuscular transmission failure. The study of muscle biopsies containing neuromuscular junctions showed well-formed post-synaptic element, synaptic acetylcholinesterase deficiency, denervation of synaptic gutters with reinnervation by terminal sprouting, and long nonmyelinated preterminal nerve segments. These data support the notion of peripheral nerve hyperexcitability in SJS, which would originate distally from synergistic actions of peripheral nerve and neuromuscular junction changes as a result of perlecan deficiency.
Subject(s)
Neuromuscular Junction/pathology , Osteochondrodysplasias/pathology , Peripheral Nerves/physiopathology , Adult , Calcium-Binding Proteins/metabolism , Electromyography , Humans , Male , Myelin Basic Protein/metabolism , Neural Conduction/physiology , Neurofilament Proteins/metabolism , Neuromuscular Junction/metabolism , Neuromuscular Junction/physiopathology , Neuromuscular Junction/ultrastructure , Peripheral Nerves/metabolism , Peripheral Nerves/pathology , Peripheral Nerves/ultrastructure , Receptor Protein-Tyrosine Kinases/metabolism , Receptor, ErbB-3/metabolism , Receptors, Cholinergic/metabolism , S100 Proteins/metabolismABSTRACT
Congenital peripheral nerve hyperexcitability (PNH) is usually associated with impaired function of voltage-gated K(+) channels (VGKCs) in neuromyotonia and demyelination in peripheral neuropathies. Schwartz-Jampel syndrome (SJS) is a form of PNH that is due to hypomorphic mutations of perlecan, the major proteoglycan of basement membranes. Schwann cell basement membrane and its cell receptors are critical for the myelination and organization of the nodes of Ranvier. We therefore studied a mouse model of SJS to determine whether a role for perlecan in these functions could account for PNH when perlecan is lacking. We revealed a role for perlecan in the longitudinal elongation and organization of myelinating Schwann cells because perlecan-deficient mice had shorter internodes, more numerous Schmidt-Lanterman incisures, and increased amounts of internodal fast VGKCs. Perlecan-deficient mice did not display demyelination events along the nerve trunk but developed dysmyelination of the preterminal segment associated with denervation processes at the neuromuscular junction. Investigating the excitability properties of the peripheral nerve suggested a persistent axonal depolarization during nerve firing in vitro, most likely due to defective K(+) homeostasis, and excluded the nerve trunk as the original site for PNH. Altogether, our data shed light on perlecan function by revealing critical roles in Schwann cell physiology and suggest that PNH in SJS originates distally from synergistic actions of peripheral nerve and neuromuscular junction changes.
Subject(s)
Axons/physiology , Heparan Sulfate Proteoglycans/physiology , Osteochondrodysplasias/pathology , Schwann Cells/physiology , Action Potentials/physiology , Aging/physiology , Animals , Basement Membrane/metabolism , Demyelinating Diseases/etiology , Disease Models, Animal , Electric Stimulation/methods , Heparan Sulfate Proteoglycans/deficiency , Heparan Sulfate Proteoglycans/genetics , Kv1.1 Potassium Channel/biosynthesis , Mice , Mice, Mutant Strains , Microscopy, Electron , Mutation , Myelin Sheath/physiology , Myelin Sheath/ultrastructure , Neuromuscular Junction/physiopathology , Osteochondrodysplasias/complications , Osteochondrodysplasias/physiopathology , Ranvier's Nodes/metabolism , Ranvier's Nodes/ultrastructure , Reverse Transcriptase Polymerase Chain Reaction/methods , Schwann Cells/metabolism , Sciatic Nerve/physiopathology , Sciatic Nerve/ultrastructureABSTRACT
Schwartz-Jampel syndrome (SJS) is a recessive neuromyotonia with chondrodysplasia. It results from hypomorphic mutations of the gene encoding perlecan, leading to a decrease in the levels of this heparan sulphate proteoglycan in basement membranes (BMs). It has been suggested that SJS neuromyotonia may result from endplate acetylcholinesterase (AChE) deficiency, but this hypothesis has never been investigated in vivo due to the lack of an animal model for neuromyotonia. We used homologous recombination to generate a knock-in mouse strain with one missense substitution, corresponding to a human familial SJS mutation (p.C1532Y), in the perlecan gene. We derived two lines, one with the p.C1532Y substitution alone and one with p.C1532Y and the selectable marker Neo, to down-regulate perlecan gene activity and to test for a dosage effect of perlecan in mammals. These two lines mimicked SJS neuromyotonia with spontaneous activity on electromyogramm (EMG). An inverse correlation between disease severity and perlecan secretion in the BMs was observed at the macroscopic and microscopic levels, consistent with a dosage effect. Endplate AChE levels were low in both lines, due to synaptic perlecan deficiency rather than major myofibre or neuromuscular junction disorganization. Studies of muscle contractile properties showed muscle fatigability at low frequencies of nerve stimulation and suggested that partial endplate AChE deficiency might contribute to SJS muscle stiffness by potentiating muscle force. However, physiological endplate AChE deficiency was not associated with spontaneous activity at rest on EMG in the diaphragm, suggesting that additional changes are required to generate such activity characteristic of SJS.
Subject(s)
Acetylcholinesterase/deficiency , Acetylcholinesterase/genetics , Isaacs Syndrome/enzymology , Isaacs Syndrome/genetics , Motor Endplate/enzymology , Osteochondrodysplasias/enzymology , Osteochondrodysplasias/genetics , Alleles , Animals , Disease Models, Animal , Electromyography , Female , Gene Dosage , Heparan Sulfate Proteoglycans/deficiency , Heparan Sulfate Proteoglycans/genetics , Humans , Isaacs Syndrome/physiopathology , Male , Mice , Mice, Inbred C57BL , Mice, Mutant Strains , Mice, Transgenic , Motor Endplate/physiopathology , Muscle Contraction/genetics , Muscle Contraction/physiology , Mutation, Missense , Osteochondrodysplasias/physiopathology , PhenotypeABSTRACT
Schwartz-Jampel syndrome (SJS) is a rare autosomal recessive condition defined by the association of myotonia with chondrodysplasia. SJS results from mutations in the HSPG2 gene, which encodes perlecan, a major component of basement membranes. Only eight HSPG2 mutations have been reported in six SJS families. Here, we describe the molecular findings in 23 families (35 patients) with SJS, being one-third of the SJS cases reported in the medical literature. We identified 22 new HSPG2 mutations and unreported polymorphisms. Mutations included nine deletion or insertion (41%), six splice site (27%), five missense (23%), and two nonsense mutations (9%). All but four mutations were private, and we found no evidence for a founder effect. Analyses of HSPG2 messenger RNA (mRNA) and perlecan immunostaining on patients' cells revealed a hypomorphic effect of the studied mutations. They also demonstrated distinct consequences of truncating and missense mutations on perlecan expression as truncating mutations resulted in instability of HSPG2 mRNA through nonsense mRNA-mediated decay, whereas missense mutations involving cysteine residues led to intracellular retention of perlecan, probably due to quality control pathways. Our analyses strengthen the idea that SJS results from hypomorphic mutations of the HSPG2 gene. They also propose tools for its molecular diagnosis and provide new clues for the understanding of its pathophysiology.
Subject(s)
Heparan Sulfate Proteoglycans/genetics , Mutation , Osteochondrodysplasias/genetics , Alternative Splicing/genetics , Base Sequence , Cells, Cultured , DNA Mutational Analysis , Female , Gene Expression , Genotype , Haplotypes , Heparan Sulfate Proteoglycans/metabolism , Humans , Male , Models, Genetic , Mutant Proteins/metabolism , Phenotype , Sequence Analysis, DNAABSTRACT
Paramyotonia congenita (PC) is a dominantly inherited skeletal muscle disorder caused by missense mutations in the SCN4A gene encoding the pore-forming alpha subunit (hSkM1) of the skeletal muscle Na+ channel. Muscle stiffness is the predominant clinical symptom. It is usually induced by exposure to cold and is aggravated by exercise. The most prevalent PC mutations occur at T1313 on DIII-DIV linker, and at R1448 on DIV-S4 of the alpha subunit. Only one substitution has been described at T1313 (T1313M), whereas four distinct amino-acid substitutions were found at R1448 (R1448C/H/P/S). We report herein a novel mutation at position 1313 (T1313A) associated with a typical phenotype of PC. We stably expressed T1313A or wild-type (hSkM1) channels in HEK293 cells, and performed a detailed study on mutant channel gating defects using the whole-cell configuration of the patch-clamp technique. T1313A mutation impaired Na+ channel fast inactivation: it slowed and reduced the voltage sensitivity of the kinetics, accelerated the recovery, and decreased the voltage-dependence of the steady state. Slow inactivation was slightly enhanced by the T1313A mutation: the voltage dependence was shifted toward hyperpolarization and its steepness was reduced compared to wild-type. Deactivation from the open state assessed by the tail current decay was only slowed at positive potentials. This may be an indirect consequence of disrupted fast inactivation. Deactivation from the inactivation state was hastened. The T1313A mutation did not modify the temperature sensitivity of the Na+ channel per se. However, gating kinetics of the mutant channels were further slowed with cooling, and reached levels that may represent the threshold for myotonia. In conclusion, our results confirm the role of T1313 residue in Na+ channel fast inactivation, and unveil subtle changes in other gating processes that may influence the clinical phenotype.
Subject(s)
Cold Temperature , Muscle, Skeletal/metabolism , Mutation, Missense , Myotonic Disorders/genetics , Myotonic Disorders/metabolism , Sodium Channels/genetics , Sodium Channels/metabolism , Adult , Alanine , Amino Acid Substitution , Cell Line , Electrophysiology , Female , Humans , Kinetics , NAV1.4 Voltage-Gated Sodium Channel , Pedigree , Phenotype , Sodium Channels/physiology , Temperature , Threonine , Time FactorsABSTRACT
SPG13, an autosomal dominant form of pure hereditary spastic paraplegia, was recently mapped to chromosome 2q24-34 in a French family. Here we present genetic data indicating that SPG13 is associated with a mutation, in the gene encoding the human mitochondrial chaperonin Hsp60, that results in the V72I substitution. A complementation assay showed that wild-type HSP60 (also known as "HSPD1"), but not HSP60 (V72I), together with the co-chaperonin HSP10 (also known as "HSPE1"), can support growth of Escherichia coli cells in which the homologous chromosomal groESgroEL chaperonin genes have been deleted. Taken together, our data strongly indicate that the V72I variation is the first disease-causing mutation that has been identified in HSP60.