Tissue informative cell-free DNA methylation sites in amyotrophic lateral sclerosis.

Cell-free DNA (cfDNA) is increasingly recognized as a promising biomarker candidate for disease monitoring. However, its utility in neurodegenerative diseases, like amyotrophic lateral sclerosis (ALS), remains underexplored. Existing biomarker discovery approaches are tailored to a specific disease context or are too expensive to be clinically practical. Here, we address these challenges through a new approach combining advances in molecular and computational technologies. First, we develop statistical tools to select tissue-informative DNA methylation sites relevant to a disease process of interest. We then employ a capture protocol to select these sites and perform targeted methylation sequencing. Multi-modal information about the DNA methylation patterns are then utilized in machine learning algorithms trained to predict disease status and disease progression. We applied our method to two independent cohorts of ALS patients and controls (n=192). Overall, we found that the targeted sites accurately predicted ALS status and replicated between cohorts. Additionally, we identified epigenetic features associated with ALS phenotypes, including disease severity. These findings highlight the potential of cfDNA as a non-invasive biomarker for ALS.

The effects of executive and behavioral dysfunction on the course of ALS.

OBJECTIVE: To determine whether patients with ALS-frontotemporal lobar dementia (FTLD) have a shorter survival and are less compliant with recommended treatments than those with ALS who have normal executive and behavioral function (classic ALS). METHODS: Survival analysis from ALS symptom onset to death included 81 of 100 consecutive patients who could be classified definitely as ALS with abnormal executive or behavioral function or as classic ALS. Criteria were defined for compliance with noninvasive positive-pressure ventilation (NPPV) and percutaneous endoscopic gastrostomy (PEG). RESULTS: Median survival was 2 years 4 months for the 28 patients with FTLD and 3 years 3 months for the 53 patients with classic ALS (relative hazard for death 1.93, CI 1.09 to 3.43; p = 0.024). However, the relative hazard associated with FTLD (1.49) in the multivariate model was diminished by the association of FTLD with bulbar onset and older age and was not significant in this sample size. With bulbar onset, median survival was 2 years 0 months for the 14 with ALS-FTLD and 2 years 10 months for the 10 with classic ALS (relative hazard for death 2.78, CI 1.02 to 7.55; p = 0.045), and older age was not a significant risk. Noncompliance with NPPV and PEG were 75% and 72% in ALS-FTLD, respectively, vs 38% and 31% in classic ALS (relative risks 2.00 and 2.34; p = 0.013 and 0.022). CONCLUSIONS: Survival is significantly shorter among patients with ALS-FTLD than with classic ALS. Furthermore, patients with ALS-FTLD are twice as likely to be noncompliant.

Comparison of family histories in FTLD subtypes and related tauopathies.

Pedigrees from 269 patients with frontotemporal lobar degeneration (FTLD), including frontotemporal dementia (FTD), FTD with ALS (FTD/ALS), progressive nonfluent aphasia, semantic dementia (SD), corticobasal degeneration, and progressive supranuclear palsy were analyzed to determine the degree of heritability of these disorders. FTD/ALS was the most and SD the least heritable subtype. FTLD syndromes appear to have different etiologies and recurrence risks.

A voxel-based morphometry study of patterns of brain atrophy in ALS and ALS/FTLD.

OBJECTIVE: To investigate the patterns of MRI brain atrophy in patients with ALS with and without clinically evident frontotemporal lobar dementia (FTLD) using voxel-based morphometry (VBM). METHODS: Voxel-based morphometry was used to compare T1-weighted MRI images obtained from ten ALS patients with FTLD, ten ALS patients who were cognitively and behaviorally normal, and 22 control subjects. Images from patients and controls were spatially pre-processed using a study-specific, customized template and a priori images. A statistical threshold of p < 0.05 corrected for multiple comparisons determined significance. RESULTS: A common pattern of gray matter atrophy was seen in both ALS and ALS/FTLD patients when compared to controls that involved the bilateral motor/premotor cortices, the left middle and inferior frontal gyri, the anterior portion of the superior frontal gyri, the superior temporal gyri, the temporal poles and left posterior thalamus. Most of the frontal regions were significantly more atrophied in the ALS/FTLD group than in the ALS group. No significant differences were found in white matter volumes. CONCLUSIONS: Patients with ALS and ALS associated with frontotemporal lobar degeneration exhibit widespread gray matter atrophy in frontotemporal regions. This finding supports the idea of a clinical and anatomic continuum between ALS and frontotemporal lobar degeneration.

A clinical trial of creatine in ALS.

BACKGROUND: Mitochondrial dysfunction occurs early in the course of ALS, and the mitochondria may be an important site for therapeutic intervention. Creatine stabilizes the mitochondrial transition pore, and is important in mitochondrial ATP production. In a transgenic mouse model of ALS, administration of creatine prolongs survival and preserves motor function and motor neurons. METHODS: The authors conducted a randomized double-blind, placebo controlled trial on 104 patients with ALS from 14 sites to evaluate the efficacy of creatine supplementation in ALS. The primary outcome measure was maximum voluntary isometric contraction of eight upper extremity muscles, with secondary outcomes including grip strength, ALS Functional Rating Scale-Revised, and motor unit number estimates. Patients were treated for 6 months, and evaluated monthly. RESULTS: Creatine was tolerated well, but no benefit of creatine could be demonstrated in any outcome measure. CI analysis showed that the study, although powered to detect a 50% or greater change in rate of decline of muscle strength, actually made an effect size of greater than 23% unlikely. It was also demonstrated that motor unit number estimation was performed with acceptable reproducibility and tolerability, and may be a useful outcome measure in future clinical trials. CONCLUSION: Any beneficial effect of creatine at 5 g per day in ALS must be small. Other agents should be considered in future studies of therapeutic agents to address mitochondrial dysfunction in ALS. In addition, motor unit number estimation may be a useful outcome measure for future clinical trials in ALS.

Are amyotrophic lateral sclerosis patients cognitively normal?

BACKGROUND: Patients with ALS are often told that the disease spares cognition; however, recent evidence suggests deficits in frontal executive skills occur in a sizable minority of ALS patients. In many instances, the frontal executive deficits represent the co-occurrence of frontotemporal lobar dementia (FTLD) and ALS. METHODS: Word generation, a simple frontal task that takes <2 minutes, was tested in 100 consecutive patients with ALS seen in the authors' multidisciplinary clinic. Any patient with a prior dementia diagnosis was excluded from the study. A subset of 44 patients agreed to undergo further neuropsychological testing and clinical interview to confirm or deny a diagnosis of dementia. RESULTS: Diminished word generation was found in one-third. Of the patients with abnormal word generation who agreed to further evaluation, nearly all were shown to meet research criteria for FTLD. In addition, one-quarter of the patients with normal word generation who agreed to further evaluation met research criteria for FTLD; these patients had new-onset personality changes. CONCLUSIONS: This study suggests that frontal executive deficits are present in half of ALS patients, many of whom meet strict research criteria for FTLD. Word generation tests are a useful screening tool in this cohort.

Effect of recording window and stimulation variables on the statistical technique of motor unit number estimation.

A variety of methods are used for the selection of recording window sizes and stimulation current levels for statistical motor unit number estimation (MUNE). This study compares different recording window sizes and stimulation current levels within those windows in the same subjects to determine the effect on MUNE value and reproducibility. Four recording windows of 10% size were compared with four of 5%, with the stimulation current set in the lower quarter, middle half, and upper quarter of the recording window. MUNE for stimulation current set in the lower quarter of the window was 81 (62-103) for 10% recording windows and 120 (108-135) for 5% recording windows, and 91 (61-123) and 133 (120-154) for stimulation current set in the middle half. Increasing the recording window size from 5 to 10% lowers the MUNE value in controls, but tends to improve reproducibility; and setting the stimulation current in the lower quarter of the window, changes the MUNE value minimally, while tending to improve further reproducibility. Excellent reproducibility of MUNE was obtained when applied to a pilot group of 10 amyotrophic lateral sclerosis patients. Based on this study, we conclude that the ideal method for statistical motor unit estimation involves using 10% recording windows and setting the stimulation current in the lower quarter of the recording window.

Comparison of multiple point and statistical motor unit number estimation.

This study compares two common techniques for motor unit number estimation, multiple point stimulation and statistical method, to determine which is more reproducible. Surface recorded motor unit action potentials (SMUPs) of the left hypothenar muscle group were measured on 20 controls and 10 ALS patients. For multiple point, 10 different threshold SMUPs were recorded. For statistical method, mean SMUP amplitude was measured at several stimulus levels, typically spanning >40% of CMAP amplitude range. Both techniques were performed twice, results averaged, electrodes changed, and all recording repeated. For controls, mean of two motor unit number estimation (MUNE) (+/- standard deviation) was 60 (+/-5) for statistical method, and 108 (+/-38) for multiple point. For ALS patients, these values were 21 (+/-16) for statistical method and 55 (+/-39) for multiple point. Test-retest correlation coefficients and coefficients of variation for mean of two MUNE were 0.98 and 7% for statistical method, and 0.90 and 12% for multiple point, respectively. Statistical method was more reproducible and faster than multiple point, supporting its utility in monitoring rates of MUNE change.

Motor unit number estimation (MUNE): how may it contribute to the diagnosis of ALS?

Motor unit number estimation (MUNE) is a type of electrophysiological technique that measures the approximate number of lower motor neurons (LMNs) innervating a single muscle or a small group of muscles. Low MUNE counts provide evidence of LMN degeneration, but a single MUNE study does not determine if this loss is ongoing, recent or remote in time. Sequential change of MUNE count provides evidence for ongoing degeneration. Furthermore, sequential change in MUNE from a normal to abnormally low count provides evidence for progressive spread of signs within a region or to another region. MUNE has no established ability to identify other diseases that may provide a non-ALS explanation for the signs of LMN degeneration. If MUNE studies were to be incorporated into a future revision of the diagnostic criteria for ALS, prospective studies will be important to define more clearly the sensitivity and specificity of MUNE in patients with ALS and in patients with weakness that does not involve LMN degeneration. In addition to its potential contributions toward the diagnosis of ALS, MUNE may have greater potential in quantifying the rate of progression in studies of the natural history of ALS and the response to experimental treatment.

Adult-onset nemaline myopathy: Another cause of dropped head.

A 59-year-old man with severe neck extensor weakness had findings diagnostic of nemaline myopathy on muscle biopsy. Review of the literature shows that dropped head occurs in nearly half of the patients with adult-onset nemaline myopathy. Other leading causes of dropped head syndrome are amyotrophic lateral sclerosis, myasthenia gravis, and isolated neck extensor myopathy.

Clinical neurophysiologic studies: which test is useful and when?

Neurophysiological studies test the integrity of nerve roots and nerves at different points from the spinal cord to the periphery. EMG is the most sensitive neurophysiological test for evaluating patients with radiculopathy, providing information on diagnosis, location, and prognosis. F-wave and H-reflex studies may be abnormal, but the information that they provide is nonspecific and usually redundant because the needle examination is abnormal anyway. Somatosensory evoked potentials are less sensitive and specific than EMG for diagnosing a radiculopathy, but uncommonly may be the only abnormality. Magnetic stimulation, paraspinal mapping, and cervical root stimulation are investigational techniques of uncertain utility.

Widespread neurotrophin receptor expression in the immune system and other nonneuronal rat tissues.

RNase protection analysis using p75, trk A, and trk B RNA probes was used to examine mRNA expression in rat tissues, with particular emphasis on the immune system. Every tissue examined, with the exception of postnatal day 0 spleen, expressed p75 mRNA. Trk A mRNA was observed in tissues previously reported to be negative for the trk A receptor, such as kidney, thymus, lymph node, muscle, and lung. Neuronal tissues expressed only the long form of trk A, whereas nonneuronal tissues expressed both trk A forms. Trk B mRNA was expressed by the same tissues as trk A, plus heart and spleen. Neuronal tissues expressed full-length and truncated trk B, whereas nonneuronal tissues only expressed truncated trk B. During development of the thymus p75 mRNA levels increased and trk A mRNA levels decreased. Similarly, for the spleen, p75 mRNA levels increased and those of trk B decreased during development. The expression of p75, trk A and trk B was localized primarily to the stroma of the thymus and spleen, but there was some expression by the splenocytes and thymocytes. The widespread expression of neurotrophin receptors in areas not known to be targets for neurotrophins suggests broader functions for neurotrophins outside of the nervous system.

Tissue-specific alternative splicing generates two isoforms of the trkA receptor.

The trkA receptor functions as a signal transducing receptor for nerve growth factor. In this report, we show that alternative splicing results in the production of two distinct trkA isoforms in both rats and humans. These isoforms differ by virtue of a 6-amino acid insertion in their extracellular domain, the placement of which corresponds exactly with the breakpoint found in several human trkA oncogenes. When tested in fibroblasts, the presence (trkAII) or absence (trkAI) of the 6-amino acid insert does not affect the receptor's ligand binding specificity or its ability to transduce functional signals in response to nerve growth factor. In rats and humans, trkAII is the only isoform expressed within neuronal tissues at appreciable levels whereas trkAI, the form of trkA originally cloned, appears to be expressed mainly in non-neuronal tissues.