Envisioning the development of a CRISPR-Cas mediated base editing strategy for a patient with a novel pathogenic CRB1 single nucleotide variant.

BACKGROUND: Inherited retinal degeneration (IRD) associated with mutations in the Crumbs homolog 1 (CRB1) gene is associated with a severe, early-onset retinal degeneration for which no therapy currently exists. Base editing, with its capability to precisely catalyse permanent nucleobase conversion in a programmable manner, represents a novel therapeutic approach to targeting this autosomal recessive IRD, for which a gene supplementation is challenging due to the need to target three different retinal CRB1 isoforms. PURPOSE: To report and classify a novel CRB1 variant and envision a possible therapeutic approach in form of base editing. METHODS: Case report. RESULTS: A 16-year-old male patient with a clinical diagnosis of early-onset retinitis pigmentosa (RP) and characteristic clinical findings of retinal thickening and coarse lamination was seen at the Oxford Eye Hospital. He was found to be compound heterozygous for two CRB1 variants: a novel pathogenic nonsense variant in exon 9, c.2885T>A (p.Leu962Ter), and a likely pathogenic missense change in exon 6, c.2056C>T (p.Arg686Cys). While a base editing strategy for c.2885T>A would encompass a CRISPR-pass mediated "read-through" of the premature stop codon, the resulting missense changes were predicted to be "possibly damaging" in in-silico analysis. On the other hand, the transversion missense change, c.2056C>T, is amenable to transition editing with an adenine base editor (ABE) fused to a SaCas9-KKH with a negligible chance of bystander edits due to an absence of additional Adenines (As) in the editing window. CONCLUSIONS: This case report records a novel pathogenic nonsense variant in CRB1 and gives an example of thinking about a base editing strategy for a patient compound heterozygous for CRB1 variants.

A novel homozygous c.67C>T variant in retinol binding protein 4 (RBP4) associated with retinitis pigmentosa and childhood acne vulgaris.

BACKGROUND: The retinol binding protein 4 (RBP4) is essential in delivering retinol to the retinal pigment epithelium and normal functioning of the visual cycle. Homozygous mutations in the RBP4 gene lead to severe retinitis pigmentosa that is phenotypically indistinguishable from retinitis pigmentosa caused by other recessive mutations. METHODS: Case Report. PURPOSE: To report a novel homozygous RBP4 c.67 C > T variant in a case of retinitis pigmentosa associated with severe childhood acne vulgaris. RESULTS: A 49-year old Caucasian man with a family history of retinitis pigmentosa, presented with low vision and night blindness from early childhood. Fundus examination showed findings typical of recessive retinitis pigmentosa. Next generation sequencing analysis revealed a novel homozygous RBP4 c.67 C > T variant. Examination of patient's back showed widespread scaring and hyperpigmentation secondary to severe childhood-onset acne vulgaris. Patient's affected brother, positive for the same homozygous variant, also had a history of severe acne vulgaris whereas the unaffected brother did not, confirming that mutations in RBP4 segregated with the acne vulgaris phenotype in this family. CONCLUSIONS: We describe a case of retinitis pigmentosa associated with acne vulgaris and highlight the role of this systemic manifestation of retinol deficiency in confirming pathogenicity of the novel variant. Given the small size of the genomic RBP4 DNA (0.6kb), gene therapy using an adeno-associated viral vector with subretinal delivery has great potential to treat this severe childhood-onset blinding retinal disease. In addition, ubiquitous expression of RBP4 supports the development of in vitro functional assays to test the vector potency for clinical use.

Novel non-contiguous exon duplication in choroideremia.

The importance of establishing a genetic diagnosis in patients with a choroideremia phenotype has been underscored by the advent of gene replacement therapy for this condition. Here, we describe a complex imbalance at the CHM locus in a male patient with classical disease. At the DNA level, this imbalance consists of 2 non-contiguous duplications (exons 1-2 and 9-12). Further characterization suggests the generation of 2 independent CHM transcriptional units, one of which may produce a deleted form of the Rab escort protein 1 protein. Expression of such a type of aberrant protein in photoreceptors may have important implications when considering gene therapy for this disorder.

CNS demyelination and quadrivalent HPV vaccination.

Vaccination is generally considered safe in patients with multiple sclerosis (MS). We report five patients who presented with multifocal or atypical demyelinating syndromes within 21 days of immunization with the quadrivalent human papilloma virus (HPV) vaccine, Gardasil. Although the target population for vaccination, young females, has an inherently high risk for MS, the temporal association with demyelinating events in these cases may be explained by the potent immuno-stimulatory properties of HPV virus-like particles which comprise the vaccine. A prospective case-control study of patients with MS or clinically isolated demyelinating syndromes receiving the Gardasil vaccine may provide relevant safety data in this population.

Driving and patients with brain tumours: a postal survey of neurosurgeons, neurologists and radiation oncologists.

This study was aimed at evaluating the consistency of driving advice given by treating clinicians to patients diagnosed with brain tumours. Secondary aims include assessing the awareness of current driving guidelines and whether or not there was a need for more specific guidelines in this group of patients. This was undertaken utilizing a scenario-based postal survey. The results show an overall poor consistency in the answers provided for each case scenario. 73.1% respondents were not aware of any current driving guidelines. Of those who were aware of driving guidelines, 67.7% wanted more specific guidelines to be developed. Possible explanations for this are a lack of awareness of the existence of any driving guidelines and a lack of objective criteria in the current driving guidelines. The authors recommend that the current driving guidelines be comprehensively distributed to clinicians who treat patients with brain tumours, as well as forming a multi-disciplinary working party to develop more specific and objective driving guidelines.

Sudomotor nerve conduction velocity and central processing time of the skin conductance response.

OBJECTIVE: To estimate the sudomotor nerve conduction velocity (CV), the central processing time (CPT) and habituation of the skin conductance response (SCR). METHODS: SCRs in response to a single deep inspiratory breath, an electrical stimulus and a sound click were obtained from the fingers and toes of 30 healthy adults. Sudomotor nerve conduction velocities were determined after measuring extremity length and latency differences. CPT was estimated by subtracting the efferent time and the known afferent times and neuroeffector times from the onset latency. RESULTS: The inspiratory SCR habituated slower than the auditory or electrical SCRs. CVs of the 3 modalities did not differ statistically and their mean was 1.07 m s(-1) (95% CI: 1.01-1.13). The inspiratory SCR arrived at the fingers 1.26+/-0.09 s after the onset of chest wall movement. Electrical and auditory SCR onset latencies at the fingers were 1.60+/-0.03 and 1.75+/-0.04 s, respectively. Their CPTs were 140 and 160 ms, estimated from the electrical and auditory SCR onset latencies to the fingers. The CPT for inspiratory SCR was estimated to occur during the inspiratory CPT after the inspiratory decision and before chest movement. CONCLUSIONS: In contrast to the SCR following an electrical or auditory stimulus, initiation of deep inspiratory SCR occurs before the inspiratory act, precluding any possible input from respiratory afferent receptors and implicating a central generator. SIGNIFICANCE: This study provides new insights into the origin of the SCR following inspiration.

The current use of botulinum toxin.

Botulinum toxin is the most potent neurotoxin known, and has been in clinical use since the late 1970s. The toxin inhibits the release of acetylcholine from nerve terminals by inhibiting transport of the synaptic vesicles, thus causing functional denervation lasting up to 6 months. Our understanding of the mechanism of action of the toxin and the spectrum of diseases treatable with this agent continues to increase. Efficacy has been demonstrated in hemifacial spasm, dystonia, spasticity, hyperhidrosis and other conditions. Alternative serotypes are used in some centres, generally after the development of immunoresistance to the standard toxin (serotype A), and are likely to be in routine use in the near future. This paper reviews the history, pharmacology and current uses of botulinum toxin.

Dynamics of SCR, EEG, and ERP activity in an oddball paradigm with short interstimulus intervals.

Studies of concurrent central, and autonomic activity using a conventional event-related potential (ERP) oddball paradigms, are considered useful in elucidating the relationship between central and autonomic responses, but the autonomic response tends to overlap. A new method was used to decompose and score overlapping skin conductance responses (SCR). This method enabled examination of dynamic relationships of phasic SCR, prestimulus electroencephalogram (EEG), and ERP to auditory target stimuli in 50 normal adults. SCR amplitude was negatively correlated to EEG and N200 amplitude. The SCR amplitude changes over time exhibited an exponential decline opposite to those of N200, alpha, and beta. All the fitted exponential functions had a time constant of 1-2 min. The findings suggest that a N200 component, active in the auditory sensory discrimination, is concomitant with the SCR. The narrow range of the time constant may provide a clue to the conjoint processes underlying central and autonomic adaptive functions.

Late components of the event-related potentials and their topography in Parkinson's disease.

Late components of the event-related potential (ERP; N100, P200, N200, and P300) were elicited using an auditory oddball paradigm (with a button-press response to target stimuli) in 15 Parkinson's disease (PD) patients and 50 normal control subjects. Compared with control subjects, PD subjects showed a significant decrease in N200 amplitude. Between-group topographical differences in N200 amplitude were evident at central (C3, Cz, C4) and temporal (T5, T3, T4, T6) regions. The results may reflect a deficit in response selection in PD possibly resulting from a dysfunction associated with the abnormalities in the central and temporal regions found to have a decreased N200 amplitude compared with normal control subjects in this study.

Dysfunctions of automatic (P300a) and controlled (P300b) processing in Parkinson's disease.

P300 Event Related Potentials components (P300a and P300b) were investigated using an auditory oddball paradigm (with a button press response to target stimuli) in 15 Parkinson's disease patients and 50 normal controls whilst simultaneously measuring electrodermal activity. Cluster analysis showed that the first 10 target stimuli generated the largest skin conductance responses. The first 10 single-trial ERP epochs were therefore analysed as an ERP sub-average for each individual. The P300a component (associated with the automatic 'Orienting Reflex') was expected to be most prevalent in this sub-average (compared with sub-averages of subsequent blocks of 10 target stimuli). Twenty-nine out of 50 normal controls (58%) elicited a P300a in the first 10 target sub-average, compared with only 2 out of 15 Parkinson's disease subjects (13%). The conventional P300b component (associated with controlled processing) was found to be significantly delayed for all sub-averages for the Parkinson's disease group when compared with controls. These preliminary findings suggest a possible dysfunction in both automatic and controlled processing in this disorder.

Diabetic neuropathy after pancreas transplantation: determinants of recovery.

Although simultaneous pancreas and kidney transplantation (SPK) achieves normoglycemia and correction of uremia in type I diabetic patients with renal failure, little data are available on long-term outcome and clinical determinants of recovery of peripheral neuropathy. In this prospective study, 219 electrophysiological studies using a standardized protocol were performed before and up to 8 years after SPK in 44 patients. Nine control diabetic recipients with functioning kidney but nonfunctioning pancreas transplants were studied on 35 occasions. Patients were 38.5+/-7.9 years old (mean+/-SD) with pretransplant diabetes present for 25.2+/-7.6 years. Significant polyneuropathy (total nerve conduction scores [NCS] <-1.0) was present in 89% before transplantation, which correlated with body weight (r=0.628, P<0.001). Two distinct patterns of neurological recovery were observed after SPK. Conduction velocity (CV) improved in a biphasic pattern, with a rapid initial recovery followed by subsequent stabilization. In contrast, the recovery of nerve amplitude was monophasic, and continued to improve for up to 8 years. Initial improvement in NCS was primarily due to an increase in CV (P=0.002 vs. baseline), and was best in shorter and younger patients. Recovery of total NCS at 6 months after SPK, assessed by multivariate analysis, was least in obese recipients and when performed in patients who had started dialysis before SPK, and was associated with lower transplant kidney isotopic glomerular filtration rate and HLA mismatch (P<0.05 to 0.001). Subsequent improvement was associated with less severe initial neuropathy, smaller body weight, and longer duration of diabetes (P<0.01 to 0.001). Fasting hyperinsulinemia was associated with impairment of initial recovery and subsequent NCS after SPK, but was worse in the control group. Recovery of nerve action potential amplitudes was predicted by better initial amplitudes and HLA mismatch, lower body weight, and the use of nifedipine (P<0.05 to 0.001). Nifedipine was used for hypertension in 33% of SPK and was associated with better CV and amplitudes, particularly in the upper limbs, where there was less neuropathy. The use of angiotensin-converting enzyme inhibitors also appeared beneficial, but this was confined to the lower limbs. SPK resulted in a gradual, sustained, and late improvement in nerve action potential amplitudes, consistent with axonal regeneration and partial reversal of diabetic neuropathy. These data suggest that early transplantation of uremic diabetic patients before onset of severe neuropathy, minimizing obesity and optimizing renal transplant function, maximizes neurological recovery after SPK. Furthermore, the preliminary data support randomized clinical trials for evaluation of nifedipine and angiotensin-converting enzyme inhibitors in diabetic neuropathy.

Transcranial cortical stimulation in disorders of the central motor pathways.

Motor evoked potentials (MEPs) were studied in 63 patients with disorders affecting central motor pathways. These were classified into five subgroups: motor neuron disease (16), multiple sclerosis (13), cerebral infarction (19), spinal cord lesions (10) and hereditary spastic paraplegia (5). Three patients with hysterical paraplegia were also studied. Results were compared to those obtained from 30 normal subjects. In normal subjects the mean central motor conduction time (CMCT) was 6.4 ms (range 3.4-8.1 ms, SD 1.0 ms) for abductor digiti minimi and 13.2 ms (range 9.7-17.0 ms, SD 2.3 ms) for tibialis anterior. Amplitude of the cortical MEPs was defined as a percentage of the size of the peripheral compound muscle action potential (CMAP) and ranged from 14 to 85%. Fifteen of 16 patients with motor neuron disease and all patients with cerebral infarction, multiple sclerosis (MS), spastic paraparesis and non-MS spinal lesions had abnormal studies including low amplitude, dispersed or absent responses and prolonged CMCTs. Patients with MS had markedly prolonged CMCTs. In three cases of hysterical weakness MEPs were within normal limits. MEPs are a useful method to detect pathology in the central motor pathways and may have a significant role in the diagnosis of disorders involving the upper motor neuron.

Automatic processing dysfunction in Parkinson's disease.

Simultaneous measures of Event Related Potentials (ERP) and electrodermal activity (EDA) allow the delineation of ERPs that did, and did not, evoke an electrodermal 'Orienting Reflex' (OR). The OR is an automatic reflex invoked by novel or significant stimuli. Our group have developed a model to quantify electrodermal OR activity acquired in conventional late component ERP paradigms with short interstimulus intervals. Target late component (N100, P200, N200, P300) ERPs (acquired in an auditory oddball paradigm) and EDA was examined in 15 Parkinson's disease (PD) subjects and 50 normal controls. Single-trial target ERPs were averaged according to whether or not they elicited an electrodermal OR. Compared with controls, the PD group showed significantly decreased N100 and N200 amplitudes in the OR related ERPs ('Orienting ERPs'). These preliminary findings suggest that conventional late component ERPs can be delineated according to whether or not they evoked an OR. The 'orienting ERPs' in PD showed more significant disturbances compared with controls, than ERPs that did not evoke an OR.

Brainstem myoclonus in a patient with non-dopa-responsive parkinsonism.

In the few reports where electrophysiologic techniques have been used to characterise stimulus-sensitive myoclonus in the setting of a parkinsonian syndrome, the origin of the myoclonus has usually been found to be cortical. We describe a patient with parkinsonism unresponsive to levodopa who had myoclonus that was both spontaneous and induced by somatosensory stimuli. In addition, autonomic symptoms and a marked sleep disturbance were present early in his illness. Results of electrophysiologic investigations including electromyography (EMG) studies, routine electroencephalography (EEG) recording, jerked locked back-averaging of EEG, and somatosensory evoked potentials were consistent with a brainstem origin for the myoclonic jerks. Following ipsilateral digital and supraorbital electrical stimulation, the earliest muscle activation occurred in the trapezius. An all-night sleep study showed frequent myoclonic jerks during sleep and markedly abnormal sleep architecture. We believe that this patient's myoclonus was related to pathologic changes in brainstem reticular nuclei that occurred as part of his disease process. To our knowledge, brainstem myoclonus has not been described as a feature of parkinsonian syndromes.

Apomorphine can increase cutaneous inhibition of motor activity in Parkinson's disease.

We studied the effect of non-nociceptive ipsilateral digital stimulation on EMG recorded from a small hand muscle before and after the administration of subcutaneous apomorphine in 6 patients with Parkinson's disease. All were receiving the drug to control ¿on-off¿ fluctuations in motor performance. Averaged rectified EMG was recorded from tonically contracted abductor pollicis brevis (APB) following index finger stimulation using a brief stimulus train. In 5 patients motor evoked potentials (MEPs) were also recorded from APB during tonic contraction. A conditioning stimulus train was applied to the index finger at intervals between 15 and 65 msec prior to the transcranial magnetic stimulus. After apomorphine administration the patient group showed a significant increase in both EMG and MEP inhibition induced by digital stimulation. In patients with Parkinson's disease who have marked motor fluctuations, the inhibitory response of upper limb motor neurones to low level digital cutaneous stimulation can be altered by dopamine agonists.

Modulation of motor activity by cutaneous input: inhibition of the magnetic motor evoked potential by digital electrical stimulation.

We examined the inhibitory effect of a brief train of digital (D2) electrical stimuli at 4 times perception threshold on transcranial magnetic motor evoked potentials (MEPs) recorded from abductor pollicis brevis (APB) and flexor carpi radialis (FCR) muscles ipsilateral to the side of D2 stimulation. We compared this to the inhibitory effect of ipsilateral D2 stimulation on averaged rectified EMG recorded at 10% maximum voluntary contraction and on F-responses and H-reflexes recorded from these same muscles. We also compared MEPs recorded following D2 stimulation just above perception threshold to MEPs following higher intensity D2 stimulation. As well, we assessed the effect of preceding D2 stimulation on MEPs recorded from a relaxed versus tonically contracted hand muscle. D2 stimulation elicited a triphasic response of modest MEP facilitation followed by inhibition and further facilitation. The duration and onset of MEP inhibition correlated with those of the initial period of rectified EMG inhibition, however, the magnitude of MEP inhibition was generally less than the magnitude of EMG inhibition, consistent with a greater inhibitory effect of digital afferents on smaller motor neurons. MEPs were not facilitated during the rebound of EMG activity (the E2 period) that usually followed the initial period of EMG inhibition (I1 period). The behavior of H-reflexes and F-responses following ipsilateral D2 stimulation suggested that inhibition of both EMG and MEPs is not mediated via presynaptic inhibition of Ia afferents, and that inhibition is augmented by descending rather than segmental input to spinal motor neurons. Tonic contraction of the target muscle during D2 stimulation decreased the inhibitory effect of the preceding digital stimulus possibly due to recruitment of larger spinal motor neurons less likely to be inhibited by cutaneous input.