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1.  A small deletion in C9orf72 hides a proportion of expansion carriers in FTLD 
Neurobiology of Aging  2015;36(3):1601.e1-1601.e5.
Frontotemporal lobar degeneration is a highly familial disease and the most common known genetic cause is the repeat expansion mutation in the gene C9orf72. We have identified 2 brothers with an expansion mutation in C9orf72 using Southern blotting that is undetectable using repeat-primed polymerase chain reaction. Sequencing using high concentrations of DNA denaturants of a bacterial artificial chromosome clone obtained from one of the brothers identified a 10-base pair deletion adjacent to the expansion that presumably confers strong secondary structure that interferes with the genotyping. Using an alternative method, we have identified missed expansion carriers in our cohort, and this number has increased by approximately 25%. This observation has important implications for patients undergoing genetic testing for C9orf72.
PMCID: PMC4353501  PMID: 25595499
Frontotemporal lobar degeneration; FTLD; C9orf72; Repeat expansion; ALS
2.  Assessing social isolation in motor neurone disease: A Rasch analysis of the MND Social Withdrawal Scale☆ 
Journal of the Neurological Sciences  2013;334(1-2):112-118.
Social withdrawal is described as the condition in which an individual experiences a desire to make social contact, but is unable to satisfy that desire. It is an important issue for patients with motor neurone disease who are likely to experience severe physical impairment. This study aims to reassess the psychometric and scaling properties of the MND Social Withdrawal Scale (MND-SWS) domains and examine the feasibility of a summary scale, by applying scale data to the Rasch model.
The MND Social Withdrawal Scale was administered to 298 patients with a diagnosis of MND, alongside the Hospital Anxiety and Depression Scale. The factor structure of the MND Social Withdrawal Scale was assessed using confirmatory factor analysis. Model fit, category threshold analysis, differential item functioning (DIF), dimensionality and local dependency were evaluated.
Factor analysis confirmed the suitability of the four-factor solution suggested by the original authors. Mokken scale analysis suggested the removal of item five. Rasch analysis removed a further three items; from the Community (one item) and Emotional (two items) withdrawal subscales. Following item reduction, each scale exhibited excellent fit to the Rasch model.
A 14-item Summary scale was shown to fit the Rasch model after subtesting the items into three subtests corresponding to the Community, Family and Emotional subscales, indicating that items from these three subscales could be summed together to create a total measure for social withdrawal.
Removal of four items from the Social Withdrawal Scale led to a four factor solution with a 14-item hierarchical Summary scale that were all unidimensional, free for DIF and well fitted to the Rasch model. The scale is reliable and allows clinicians and researchers to measure social withdrawal in MND along a unidimensional construct.
PMCID: PMC3837185  PMID: 24011605
Social withdrawal; Rasch analysis; Psychological distress; ALS; Amyotrophic lateral sclerosis; MND; Motor neurone disease
3.  The m.3291T>C mt-tRNALeu(UUR) mutation is definitely pathogenic and causes multisystem mitochondrial disease 
Journal of the Neurological Sciences  2013;325(1-2):165-169.
Mitochondrial tRNA point mutations are important causes of human disease, and have been associated with a diverse range of clinical phenotypes. Definitively proving the pathogenicity of any given mt-tRNA mutation requires combined molecular, genetic and functional studies. Subsequent evaluation of the mutation using a pathogenicity scoring system is often very helpful in concluding whether or not the mutation is causing disease. Despite several independent reports linking the m.3291T>C mutation to disease in humans, albeit in association with several different phenotypes, its pathogenicity remains controversial. A lack of conclusive functional evidence and an over-emphasis on the poor evolutionary conservation of the affected nucleotide have contributed to this controversy. Here we describe an adult patient who presented with deafness and lipomas and evidence of mitochondrial abnormalities in his muscle biopsy, who harbours the m.3291T > C mutation, providing conclusive evidence of pathogenicity through analysis of mutation segregation with cytochrome c oxidase (COX) deficiency in single muscle fibres, underlining the importance of performing functional studies when assessing pathogenicity.
PMCID: PMC3560033  PMID: 23273904
Mitochondrial DNA disease; Lipoma; mt-tRNALeu(UUR); Evolutionary conservation; Single-fibre studies; Pathogenicity
4.  Protocol for diaphragm pacing in patients with respiratory muscle weakness due to motor neurone disease (DiPALS): a randomised controlled trial 
BMC Neurology  2012;12:74.
Motor neurone disease (MND) is a devastating illness which leads to muscle weakness and death, usually within 2-3 years of symptom onset. Respiratory insufficiency is a common cause of morbidity, particularly in later stages of MND and respiratory complications are the leading cause of mortality in MND patients. Non Invasive Ventilation (NIV) is the current standard therapy to manage respiratory insufficiency. Some MND patients however do not tolerate NIV due to a number of issues including mask interface problems and claustrophobia. In those that do tolerate NIV, eventually respiratory muscle weakness will progress to a point at which intermittent/overnight NIV is ineffective. The NeuRx RA/4 Diaphragm Pacing System was originally developed for patients with respiratory insufficiency and diaphragm paralysis secondary to stable high spinal cord injuries. The DiPALS study will assess the effect of diaphragm pacing (DP) when used to treat patients with MND and respiratory insufficiency.
108 patients will be recruited to the study at 5 sites in the UK. Patients will be randomised to either receive NIV (current standard care) or receive DP in addition to NIV. Study participants will be required to complete outcome measures at 5 follow up time points (2, 3, 6, 9 and 12 months) plus an additional surgery and 1 week post operative visit for those in the DP group. 12 patients (and their carers) from the DP group will also be asked to complete 2 qualitative interviews.
The primary objective of this trial will be to evaluate the effect of Diaphragm Pacing (DP) on survival over the study duration in patients with MND with respiratory muscle weakness. The project is funded by the National Institute for Health Research, Health Technology Assessment (HTA) Programme (project number 09/55/33) and the Motor Neurone Disease Association and the Henry Smith Charity. Trial Registration: Current controlled trials ISRCTN53817913. The views and opinions expressed therein are those of the authors and do not necessarily reflect those of the HTA programme, NIHR, NHS or the Department of Health.
PMCID: PMC3462709  PMID: 22897892
5.  Development of a patient reported outcome measure for fatigue in motor neurone disease: the Neurological Fatigue Index (NFI-MND) 
The objective of this research was to develop a disease-specific measure for fatigue in patients with motor neurone disease (MND) by generating data that would fit the Rasch measurement model. Fatigue was defined as reversible motor weakness and whole-body tiredness that was predominantly brought on by muscular exertion and was partially relieved by rest.
Qualitative interviews were undertaken to confirm the suitability of a previously identified set of 52 neurological fatigue items as relevant to patients with MND. Patients were recruited from five U.K. MND clinics. Questionnaires were administered during clinic or by post. A sub-sample of patients completed the questionnaire again after 2-4 weeks to assess test-retest validity. Exploratory factor analyses and Rasch analysis were conducted on the item set.
Qualitative interviews with ten MND patients confirmed the suitability of 52 previously identified neurological fatigue items as relevant to patients with MND. 298 patients consented to completing the initial questionnaire including this item set, with an additional 78 patients completing the questionnaire a second time after 4-6 weeks. Exploratory Factor Analysis identified five potential subscales that could be conceptualised as representing: 'Energy', 'Reversible muscular weakness' (shortened to 'Weakness'), 'Concentration', 'Effects of heat' and 'Rest'. Of the original five factors, two factors 'Energy' and 'Weakness' met the expectations of the Rasch model. A higher order fatigue summary scale, consisting of items from the 'Energy' and 'Weakness' subscales, was found to fit the Rasch model and have acceptable unidimensionality. The two scales and the higher order summary scale were shown to fulfil model expectations, including assumptions of unidimensionality, local independency and an absence of differential item functioning.
The Neurological Fatigue Index for MND (NFI-MND) is a simple, easy-to-administer fatigue scale. It consists of an 8-item fatigue summary scale in addition to separate scales for measuring fatigue experienced as reversible muscular weakness and fatigue expressed as feelings of low energy and whole body tiredness. The underlying two factor structure supports the patient concept of fatigue derived from qualitative interviews in this population. All three scales were shown to be reliable and capable of interval level measurement.
PMCID: PMC3282643  PMID: 22107756
6.  Rasch analysis of the hospital anxiety and depression scale (hads) for use in motor neurone disease 
The Hospital Anxiety and Depression Scale (HADS) is commonly used to assess symptoms of anxiety and depression in motor neurone disease (MND). The measure has never been specifically validated for use within this population, despite questions raised about the scale's validity. This study seeks to analyse the construct validity of the HADS in MND by fitting its data to the Rasch model.
The scale was administered to 298 patients with MND. Scale assessment included model fit, differential item functioning (DIF), unidimensionality, local dependency and category threshold analysis.
Rasch analyses were carried out on the HADS total score as well as depression and anxiety subscales (HADS-T, D and A respectively). After removing one item from both of the seven item scales, it was possible to produce modified HADS-A and HADS-D scales which fit the Rasch model. An 11-item higher-order HADS-T total scale was found to fit the Rasch model following the removal of one further item.
Our results suggest that a modified HADS-A and HADS-D are unidimensional, free of DIF and have good fit to the Rasch model in this population. As such they are suitable for use in MND clinics or research. The use of the modified HADS-T as a higher-order measure of psychological distress was supported by our data. Revised cut-off points are given for the modified HADS-A and HADS-D subscales.
PMCID: PMC3192662  PMID: 21955749
7.  Selective DNAzyme-mediated cleavage of AChR mutant transcripts by targeting the mutation site or through mismatches in the binding arm 
Many dominantly inherited disorders are caused by missense amino acid substitutions resulting from a single nucleotide exchange in the encoding gene. For these disorders, where proteins expressed from the mutant alleles are often pathogenic and present throughout life, gene silencing, through intervention at the mRNA level, holds promise as a therapeutic approach. We have used mutations that underlie the slow channel congenital myasthenic syndrome (SCCMS) as a model system to study allele-specific gene silencing of RNA transcripts by DNAzymes. We tested the ability of DNAzymes to give allele-specific cleavage for i) mutations that create cleavage sites, and ii) mutations located close to a DNAzyme cleavage site that create a potential mismatch in the binding arms. For both we demonstrate selective cleavage of mutant transcripts under simulated physiological conditions. For DNAzymes with binding arm mismatches the degree of selectivity for mutant over wild type may be enhanced by optimising the mismatch position as well as the binding arm length. The optimal sites for mismatches are 1.1 and 1.2 in arm I, and 16.2 in arm II. Asymmetric binding arm DNAzymes with a shorter arm I are more discriminative. Our results show it should be possible to apply DNAzyme-mediated cleavage of mutant alleles even when the mutant does not itself create a putative cleavage site. This therapeutic approach may be well suited to dominantly inherited disorders such as SCCMS, where loss of some wild type transcripts is unlikely to have pathogenic consequences.
PMCID: PMC2737196  PMID: 19771202
DNAzyme; allele-specific silencing; binding arm mismatch; slow channel myasthenic syndrome; gene therapy
8.  Selective cleavage of AChR cRNAs harbouring mutations underlying the slow channel myasthenic syndrome by hammerhead ribozymes 
Slow channel congenital myasthenic syndrome (SCCMS) is a dominant disorder caused by missense mutations in muscle acetylcholine receptors (AChR). Expression from mutant alleles causes prolonged AChR ion-channel activations. This ‘gain of function’ results in excitotoxic damage due to excess entry of calcium ions that manifests as an endplate myopathy. The biology of SCCMS provides a model system to investigate the potential of catalytic nucleic acids for therapy in dominantly inherited disorders involving single missense mutations. Hammerhead ribozymes can catalytically cleave RNA transcripts in a sequence-specific manner. We designed hammerhead ribozymes to target transcripts from four SCCMS mutations, αT254I, αS226F, αS269I and εL221F. Ribozymes were incubated with cRNA transcripts encoding wild type and mutant AChR subunits. The ribozymes efficiently cleaved the mutant allele cRNA transcripts but left the wild type cRNA intact. Cleavage efficiency was optimised for αS226F. We were able to demonstrate robust catalytic activity under simulated physiological conditions and at high Ca2+ concentrations, which is likely to be accumulated at the endplate region of the SCCMS patient muscles. These results demonstrate the potential for gene therapy applications of ribozymes to specifically down-regulate expression of mutant alleles in dominantly inherited disorders.
PMCID: PMC2737193  PMID: 19771201
Slow channel myasthenic syndrome; hammerhead ribozymes; AChR; gene therapy; allele-specific mRNA cleavage

Results 1-8 (8)