Improved PCR based methods for detecting C9orf72 hexanucleotide repeat expansions

Due to the GC-rich, repetitive nature of C9orf72 hexanucleotide repeat expansions, PCR based detection methods are challenging. Several limitations of PCR have been reported and overcoming these could help to define the pathogenic range. There is also a need to develop improved repeat-primed PCR assays which allow detection even in the presence of genomic variation around the repeat region. We have optimised PCR conditions for the C9orf72 hexanucleotide repeat expansion, using betaine as a co-solvent and specific cycling conditions, including slow ramping and a high denaturation temperature. We have developed a flanking assay, and repeat-primed PCR assays for both 3′ and 5′ ends of the repeat expansion, which when used together provide a robust strategy for detecting the presence or absence of expansions greater than ∼100 repeats, even in the presence of genomic variability at the 3′ end of the repeat. Using our assays, we have detected repeat expansions in 47/442 Scottish ALS patients. Furthermore, we recommend the combined use of these assays in a clinical diagnostic setting

Genotype-phenotype characterisation of long survivors with motor neuron disease in Scotland

Background: We investigated the phenotypes and genotypes of a cohort of ‘long-surviving’ individuals with motor neuron disease (MND) to identify potential targets for prognostication. Methods: Patients were recruited via the Clinical Audit Research and Evaluation for MND (CARE-MND) platform, which hosts the Scottish MND Register. Long survival was defined as > 8 years from diagnosis. 11 phenotypic variables were analysed. Whole genome sequencing (WGS) was performed and variants within 49 MND-associated genes examined. Each individual was screened for C9orf72 repeat expansions. Data from ancestry-matched Scottish populations (the Lothian Birth Cohorts) were used as controls. Results: 58 long survivors were identified. Median survival from diagnosis was 15.5 years. Long survivors were significantly younger at onset and diagnosis than incident patients and had a significantly longer diagnostic delay. 42% had the MND subtype of primary lateral sclerosis (PLS). WGS was performed in 46 individuals: 14 (30.4%) had a potentially pathogenic variant. 4 carried the known SOD1 p.(Ile114Thr) variant. Significant variants in FIG4, hnRNPA2B1, SETX, SQSTM1, TAF15, and VAPB were detected. 2 individuals had a variant in the SPAST gene suggesting phenotypic overlap with hereditary spastic paraplegia (HSP). No long survivors had pathogenic C9orf72 repeat expansions. Conclusions: Long survivors are characterised by younger age at onset, increased prevalence of PLS and longer diagnostic delay. Genetic analysis in this cohort has improved our understanding of the phenotypes associated with the SOD1 variant p.(Ile114Thr). Our findings confirm that pathogenic expansion of C9orf72 is likely a poor prognostic marker. Genetic screening using targeted MND and/or HSP panels should be considered in those with long survival, or early-onset slowly progressive disease, to improve diagnostic accuracy and aid prognostication

Mutations in the SPG7 gene cause chronic progressive external ophthalmoplegia through disordered mitochondrial DNA maintenance.

Despite being a canonical presenting feature of mitochondrial disease, the genetic basis of progressive external ophthalmoplegia remains unknown in a large proportion of patients. Here we show that mutations in SPG7 are a novel cause of progressive external ophthalmoplegia associated with multiple mitochondrial DNA deletions. After excluding known causes, whole exome sequencing, targeted Sanger sequencing and multiplex ligation-dependent probe amplification analysis were used to study 68 adult patients with progressive external ophthalmoplegia either with or without multiple mitochondrial DNA deletions in skeletal muscle. Nine patients (eight probands) were found to carry compound heterozygous SPG7 mutations, including three novel mutations: two missense mutations c.2221G>A; p.(Glu741Lys), c.2224G>A; p.(Asp742Asn), a truncating mutation c.861dupT; p.Asn288*, and seven previously reported mutations. We identified a further six patients with single heterozygous mutations in SPG7, including two further novel mutations: c.184-3C>T (predicted to remove a splice site before exon 2) and c.1067C>T; p.(Thr356Met). The clinical phenotype typically developed in mid-adult life with either progressive external ophthalmoplegia/ptosis and spastic ataxia, or a progressive ataxic disorder. Dysphagia and proximal myopathy were common, but urinary symptoms were rare, despite the spasticity. Functional studies included transcript analysis, proteomics, mitochondrial network analysis, single fibre mitochondrial DNA analysis and deep re-sequencing of mitochondrial DNA. SPG7 mutations caused increased mitochondrial biogenesis in patient muscle, and mitochondrial fusion in patient fibroblasts associated with the clonal expansion of mitochondrial DNA mutations. In conclusion, the SPG7 gene should be screened in patients in whom a disorder of mitochondrial DNA maintenance is suspected when spastic ataxia is prominent. The complex neurological phenotype is likely a result of the clonal expansion of secondary mitochondrial DNA mutations modulating the phenotype, driven by compensatory mitochondrial biogenesis

Clinical trials in amyotrophic lateral sclerosis:a systematic review and perspective

Amyotrophic lateral sclerosis is a progressive and devastating neurodegenerative disease. Despite decades of clinical trials, effective disease modifying drugs remain scarce. To understand the challenges of trial design and delivery, we performed a systematic review of phase II, phase II/III and phase III amyotrophic lateral sclerosis clinical drug trials on trial registries and PubMed between 2008 and 2019. We identified 125 trials, investigating 76 drugs and recruiting more than 15000 people with amyotrophic lateral sclerosis. 90% of trials used traditional fixed designs. The limitations in understanding of disease biology, outcome measures, resources and barriers to trial participation in a rapidly progressive, disabling and heterogenous disease hindered timely and definitive evaluation of drugs in two-arm trials. Innovative trial designs, especially adaptive platform trials may offer significant efficiency gains to this end. We propose a flexible and scalable multi-arm, multi-stage trial platform where opportunities to participate in a clinical trial can become the default for people with amyotrophic lateral sclerosis

Genetic epidemiology of motor neuron disease-associated variants in the Scottish population

Genetic understanding of motor neuron disease (MND) has evolved greatly in the past 10 years, including the recent identification of association between MND and variants in TBK1 and NEK1. Our aim was to determine the frequency of pathogenic variants in known MND genes and to assess whether variants in TBK1 and NEK1 contribute to the burden of MND in the Scottish population. SOD1, TARDBP, OPTN, TBK1, and NEK1 were sequenced in 441 cases and 400 controls. In addition to 44 cases known to carry a C9orf72 hexanucleotide repeat expansion, we identified 31 cases and 2 controls that carried a loss-of-function or pathogenic variant. Loss-of-function variants were found in TBK1 in 3 cases and no controls and, separately, in NEK1 in 3 cases and no controls. This study provides an accurate description of the genetic epidemiology of MND in Scotland and provides support for the contribution of both TBK1 and NEK1 to MND susceptibility in the Scottish population

The development and application of the Semliki Forest virus expression system to the study of GABAA receptors

GABAA receptors are presumed to be pentameric hetero oloigomers composed of subunits potentially drawn from five subunit classes with multiple members; α(1-6), β (1-4), [gamma] (1-3), δ, and ԑ. The mechanisms by which neurones assemble this potentially large number of subunits into functional GABAA receptors is unknown. Heterologous expression systems have demonstrated that particular combinations of GABAA subunits assemble to form channels whilst others are selectively retained within the endoplasmic reticulum of the cell. In order to determine whether the assembly rules which have been defined in such heterologous expression system are representative of the assembly of GABAA receptor subunits within primary cultures of neurones, a viral expression system was used to express recombinant epitope tagged GABAA subunits in neurones. The viral expression system which was chosen was based upon the Semliki Forest virus. This particular viral expression system was chosen because of its ability to infect neurones as well as many other cell types with high efficiency. Recombinant viral particles were produced which could direct the synthesis of the α1 (9E10), β2 (9E10), [gamma]2 L FLAG and [gamma]2 S (9E10) tagged subunits. Using the Semliki viral expression system the assembly of αl (9E10) and β2 (9E10) subunits in BHK cells was compared to that produced when these subunits were transiently transfected into HEK 293 cells. The results of the viral infections replicated the assembly work performed using conventional transient transfections of HEK 293 cells. Expression of individual αl (9E10) or β2 (9E10) subunits resulted in their selective retention within the endoplasmic reticulum of the cells whilst on co-expression both the αl (9E10) and β2 (9E10) subunits could access the cell surface. Whole cell electron microscopy of such cells demonstrated that the subunits were on the surface of the cell and were preferentially associated with the microvilli of the BHK cells. The powerful nature of the Semliki viral expression system allowed the demonstration of a biophysical association between α1 (9E10) and β2 (9E10) proteins as demonstrated by changes in the sedimentation coefficients of the two proteins on co-infection of BHK cells by the two viral species. When individually expressed the α1 (9E10) and β2 (9E10) proteins have 5S sedimentation coefficients. On co-infection the sedimentation coefficients of both proteins shifted to 9S. For the first time, the viral expression system enabled the efficiency of the assembly of the α1 (9E10) and β2 (9E10) subunits to be estimated. It also provided a means to study the stability of assembled and unassembled receptor composed of α1 (9E10) and β2 (9E10) subunits. It was found that only 33% of the available α1 (9E10) and β2 (9E10) subunits became incorporated into the assembled 9S complex. Once formed, this 9S sedimenting species was relatively stable only dropping 10[percent] of its initial signal over a 16 hour period. In contrast the unassembled receptor was quickly degraded, with a 75[percent] drop over 6 hours. The half life of individually virally expressed α1 (9E10) and β2 (9E10) subunits was also investigated and determined to be between 2 to 3 hours. The stoichiometry of a GABAA receptor composed of α1 (9E10) and β2 (9E10) receptors was also evaluated using the Semliki expression system. Metabolic labelling in combination with 9E10 immunoprecipitaiton of the assembled 9S receptor allowed a determination of the ratio of α to β subunits after correction for the relative methionine contents of the two proteins. The ratio of α1 (9E10) to β2 (9E10) subunits was 1:1. The toxicity of viral infection upon SCG neurones was studied by infecting SCG neurones with a recombinant SFV virus engineered to make the Green Fluorescent Protein and then subsequently recording the resting membrane potential of neurones which could produce green fluorescence. The neurones which produced green fluorescence had normal resting potentials and a normal ability to produce action potentials. Knowing that the viral expression system had limited toxicity to the SCG neurones and that the expression system was capable of supporting the assembly of the α1 (9E10) and β2 (9E10) subunits within BHK cells the assembly of these subunits was studied in primary cultures of SCG neurones. Infection of such cultures by either the α1 (9E10) or β2 (9E10) virus particles resulted in the retention of the GABAA subunit within the cell body of the neurone. On co-infection marked surface expression could be demonstrated. This indicated that a common assembly mechanism occurs between neurones and transformed cell lines, with retention within the endoplasmic reticulum and subsequent degradation featuring as a major control mechanism. The biochemistry, and cell biology of the [gamma]2 subunit was also investigated using the Semliki expression system. The [gamma]2 S (9E10) protein was demonstrated to behave differently to the long isoform on polyacrylamide gel electrophoresis (PAGE) and sucrose gradient sedimentation studies. The size of the long isoform on PAGE was 45KDa which contrasted with the 50KDa size for the [gamma]2 S (9E10). The sucrose gradient studies indicated that the [gamma]2 S (9E10) protein had a sharp profile peaking at 3S whilst the [gamma]2 L FLAG had a much broader profile with its peak nearer 4S and aggregates between 5 and 7S. Viral expression of the short and long splice variants of the [gamma]2 subunit within BHK cells and SCG neurones produced contrasting cell biology. The short variant was shown to be capable of trafficking to the surface of BHK cells and into the processes of SCG neurones. The short splice variant was also capable of being endocytosed from the surface of BHK cells. Electron microscopic examination of BHK cells infected with the [gamma]2 S (9E10) virus was used to demonstrate the association of the [gamma]2 S (9E10) protein with coated pits. In contrast the long variant was not able to access the plasma membrane when individually expressed. Expression of the long isoform of the [gamma]2 subunit within SCG neurones produced only a cell body pattern of expression consistent with the retention of the subunit within the endoplasmic reticulum. Finally the capability of the [gamma]2 subunit to be tyrosine phosphorylated within cells expressing high levels of the tyrosine kinase Src was tested. When transiently coexpressed with α 1, β 1 and v-Src both the [gamma]2 L (9E10) and [gamma]2 S (9E10) proteins could be shown to undergo phosphorylation specifically at tyrosine residue 365 and 367 in the short and 373 and 375 in the long