Nicholas A. Fletcher

Next generation sequencing for molecular diagnosis of neurological disorders using ataxias as a model

Many neurological conditions are caused by immensely heterogeneous gene mutations. The diagnostic process is often long and complex with most patients undergoing multiple invasive and costly investigations without ever reaching a conclusive molecular diagnosis. The advent of massively parallel, next-generation sequencing promises to revolutionize genetic testing and shorten the ‘diagnostic odyssey’ for many of these patients. We performed a pilot study using heterogeneous ataxias as a model neurogenetic disorder to assess the introduction of next-generation sequencing into clinical practice. We captured 58 known human ataxia genes followed by Illumina Next-Generation Sequencing in 50 highly heterogeneous patients with ataxia who had been extensively investigated and were refractory to diagnosis. All cases had been tested for spinocerebellar ataxia 1–3, 6, 7 and Friedrich’s ataxia and had multiple other biochemical, genetic and invasive tests. In those cases where we identified the genetic mutation, we determined the time to diagnosis. Pathogenicity was assessed using a bioinformatics pipeline and novel variants were validated using functional experiments. The overall detection rate in our heterogeneous cohort was 18% and varied from 8.3% in those with an adult onset progressive disorder to 40% in those with a childhood or adolescent onset progressive disorder. The highest detection rate was in those with an adolescent onset and a family history (75%). The majority of cases with detectable mutations had a childhood onset but most are now adults, reflecting the long delay in diagnosis. The delays were primarily related to lack of easily available clinical testing, but other factors included the presence of atypical phenotypes and the use of indirect testing. In the cases where we made an eventual diagnosis, the delay was 3–35 years (mean 18.1 years). Alignment and coverage metrics indicated that the capture and sequencing was highly efficient and the consumable cost was ∼£400 (€460 or US

A 10 year retrospective audit of long-term apomorphine use in Parkinson's disease.

620). Our pathogenicity interpretation pathway predicted 13 different mutations in eight different genes: PRKCG, TTBK2, SETX, SPTBN2, SACS, MRE11, KCNC3 and DARS2 of which nine were novel including one causing a newly described recessive ataxia syndrome. Genetic testing using targeted capture followed by next-generation sequencing was efficient, cost-effective, and enabled a molecular diagnosis in many refractory cases. A specific challenge of next-generation sequencing data is pathogenicity interpretation, but functional analysis confirmed the pathogenicity of novel variants showing that the pipeline was robust. Our results have broad implications for clinical neurology practice and the approach to diagnostic testing.

Non-subtype-selective opioid receptor antagonism in treatment of levodopa-induced motor complications in Parkinson's disease

ObjectivesApomorphine is a potent dopamine agonist useful in the treatment of Parkinson’s disease patients with disabling motor fluctuations and ‘off’ periods, not responding to oral medication. It can also be of benefit in reducing dyskinesia by providing more constant dopaminergic stimulation and permitting lower levodopa dosage. However, there is a paucity of information on long-term benefits of apomorphine, including no large-scale phase III trial. We have examined our experience of apomorphine over the last 10 years, to assess indications, pattern of use, efficacy, and side effect profile.MethodsAll patients requiring apomorphine were identified through the Parkinson’s disease Nurse Specialist’s records. An audit form was produced so that the same information was gathered from all case-notes.ResultsThere were 107 patients (61 males and 46 females). Mean age of disease onset was 50.9 years, SD±9.3 (range 29–78). The mean duration of disease at start of apomorphine treatment was 10 years (SD±4.8, range2–29). The most common indications for apomorphine were severe unpredictable ‘off’ periods (75.7 %), motor fluctuations (18.7 %) and dyskinesia (5.6 %). Most patients (63.6 %) used both intermittent subcutaneous injections and infusion via pump; 25.2% were on intermittent injection, and 11.2 % infusion alone. Mean dose per injection was 3.7mg. Mean infusion dose 69.8mg, running over a mean of 13.5 hours. The mean duration of intermittent apomorphine use was 48.2 months. The mean duration of infusion was 25.1 months. Complications included skin problems in 16 patients, 2 had symptomatic hypotension, 2 worsening confusion, 1 new confusion and 5 new hallucinations (after sometime on apomorphine). Sixteen patients have stopped using apomorphine completely. Thirteen have stopped the pump, but continue on intermittent injections.ConclusionSubcutaneous apomorphine is easy for patients to use, is well tolerated and has a low incidence of side effects, especially confusion.

Use of the NeuroMate Stereotactic Robot in a Frameless Mode for Movement Disorder Surgery

Opioid peptide transmission is enhanced in the striatum of animal models and Parkinsons disease (PD) patients with levodopa‐induced motor complications. Opioid receptor antagonists reduce levodopa‐induced dyskinesia in primate models of PD; however, clinical trials to date have been inconclusive. A double‐blind, placebo controlled, crossover design study in 14 patients with PD experiencing motor fluctuations was carried out, using the non‐subtype‐selective opioid receptor antagonist naloxone. Naloxone did not reduce levodopa‐induced dyskinesia. The duration of action of levodopa was increased significantly by 17.5%. Non‐subtype‐selective opioid receptor antagonism may prove useful in the treatment of levodopa‐related wearing‐off in PD but not in dyskinesia.

Variability in position of the subthalamic nucleus targeted by magnetic resonance imaging and microelectrode recordings as compared to atlas co-ordinates.

Background/Aims: To evaluate the use of the NeuroMate stereotactic robot with a novel ultrasound registration system for movement disorder surgery (MDS). Methods: Using the robot in a frameless mode, 51 patients underwent MDS. Surgical planning was carried out using MRI data obtained more than 24 h before surgery. Results: 37 out of 50 targets in the subthalamic nucleus were satisfactorily identified with a single microelectrode trajectory and the final electrode positions were at a mean distance of 1.7 mm from the calculated target. There was a significant improvement in motor scores of the Unified Parkinson’s Disease Rating Scale III (off medication) at 6 (43%) and 18 months (51.7%) compared to pre-operative scores (p < 0.05). Conclusions: The frameless robot using only MRI data can be used for MDS. The temporal separation of imaging from the surgical procedure provides additional time for detailed image analysis and planning.

Deep brain stimulation of the subthalamic nucleus: effectiveness in advanced Parkinson’s disease patients previously reliant on apomorphine

Background: Traditional methods for localisation of target nuclei for deep brain stimulation (DBS) have used brain atlas co-ordinates for initial targeting. It is now possible to visualise the subthalamic nucleus (STN) on magnetic resonance imaging (MRI) and determine the individual variability of its position. Methods: The present study was performed in patients undergoing STN DBS for Parkinson’s disease. The STN was directly targeted from axially obtained MRI and verified with microelectrode recordings. Postoperatively, the most effective contact was identified for each patient, and its position was calculated. Results: Fifty electrodes were inserted in 25 patients. The target position varied considerably in relation to the mid-commissural point. The mean effective contact position lies just dorsal to the location of the STN in a standard brain atlas. Conclusion: The STN varies in position, and can be accurately targeted from MRI alone.

Variably protease-sensitive prionopathy in the UK: a retrospective review 1991–2008

Objectives: To assess the efficacy of bilateral subthalamic nucleus (STN) deep brain stimulation (DBS) in patients with advanced Parkinson’s disease previously reliant on apomorphine as their main antiparkinsonian medication. Methods: Seven patients with motor fluctuations despite optimal medical treatment given as predominantly apomorphine infusion (n=6), or intermittent apomorphine injections (n=1) underwent bilateral STN DBS using frameless stereotactic surgery. Standard assessments of parkinsonism and motor fluctuations, using Unified Parkinson’s Disease Rating Scale (UPDRS) were performed before and six months after surgery. Assessments were performed both on and off medication, and postoperative with the stimulators switched on and off. Results: Bilateral STN DBS improved motor scores (UPDRS III) by 61% when off medication (p<0.05). Clinical fluctuations (UPDRS IV items 36–39) were reduced by 46.2% (p<0.05). Total daily apomorphine dose was reduced by 68.9% (p<0.05) and apomorphine infusion via a pump was no longer required in four patients. There were no operative complications. Two patients required treatment for hallucinations postoperatively but there was no significant change in mini-mental state examination. Conclusions: In patients with advanced Parkinson’s disease, previously reliant on apomorphine, bilateral STN DBS is an effective treatment to reduce motor fluctuations and enable a reduction in apomorphine use.

Cannabis-induced propriospinal myoclonus

Variably protease-sensitive prionopathy is a newly described human prion disease of unknown aetiology lying out with the hitherto recognized phenotypic spectrum of Creutzfeldt-Jakob disease. Two cases that conform to the variably protease-sensitive prionopathy phenotype have been identified prospectively in the U.K. since the first description of the condition in 2008 in the U.S.A. To determine the incidence and phenotype of variably protease-sensitive prionopathy within a single well-defined cohort, we have conducted a retrospective review of patients referred to the National Creutzfeldt-Jakob Disease Research & Surveillance Unit during the period 1991-2008. The approach taken was to screen frozen brain tissue by western blotting for the form of protease-resistant prion protein that characterizes variably protease-sensitive prionopathy, followed by neuropathological and clinical review of candidate cases. Cases diagnosed as sporadic Creutzfeldt-Jakob disease with atypical neuropathology were also reviewed. Four hundred and sixty-five cases were screened biochemically, yielding four candidate cases of variably protease-sensitive prionopathy. One was discounted on pathological and clinical grounds, and one was a known case of variably protease-sensitive prionopathy previously reported, leaving two new cases, which were confirmed biochemically and neuropathologically as variably protease-sensitive prionopathy. A third new case that lacked frozen tissue was recognized retrospectively on neuropathological grounds alone. This means that five cases of variably protease-sensitive prionopathy have been identified (prospectively and retrospectively) during the surveillance period 1991-2011 in the U.K. Assuming ascertainment levels equivalent to that of other human prion diseases, these data indicate that variably protease-sensitive prionopathy is a rare phenotype within human prion diseases, which are themselves rare. Biochemical investigation indicates that the abnormal protease-resistant prion protein fragment that characterizes variably protease-sensitive prionopathy is detectable at low levels in some cases of sporadic Creutzfeldt-Jakob disease and conversely, that the form of abnormal prion protein that characterizes sporadic Creutzfeldt-Jakob disease can be found in certain brain regions of cases of variably protease-sensitive prionopathy, indicating molecular overlaps between these two disorders.

Paraparesis Due to Syphilitic Aortic Dissection

We report on a case of a 25‐year‐old woman with clusters of myoclonus induced by a single exposure to inhaled cannabis. Investigations excluded a structural abnormality of the spine. Multi‐channel surface EMG with parallel frontal EEG recording confirmed the diagnosis of propriospinal myoclonus.

Myelopathy but normal MRI: where next?

A patient is presented who developed an acute spinal cord infarct with paraparesis and segmental pain but no sensory or sphincter involvement.Investigations revealed an extensive dissecting thoracic aortic aneurysm and positive treponemal serologic findings consistent with tertiary cardiovascular syphilis. NEUROLOGY 1997;48: 221-223