Susana Pinto

Haploinsufficiency of TBK1 causes familial ALS and fronto-temporal dementia

Amyotrophic lateral sclerosis (ALS) is a genetically heterogeneous neurodegenerative syndrome hallmarked by adult-onset loss of motor neurons. We performed exome sequencing of 252 familial ALS (fALS) and 827 control individuals. Gene-based rare variant analysis identified an exome-wide significant enrichment of eight loss-of-function (LoF) mutations in TBK1 (encoding TANK-binding kinase 1) in 13 fALS pedigrees. No enrichment of LoF mutations was observed in a targeted mutation screen of 1,010 sporadic ALS and 650 additional control individuals. Linkage analysis in four families gave an aggregate LOD score of 4.6. In vitro experiments confirmed the loss of expression of TBK1 LoF mutant alleles, or loss of interaction of the C-terminal TBK1 coiled-coil domain (CCD2) mutants with the TBK1 adaptor protein optineurin, which has been shown to be involved in ALS pathogenesis. We conclude that haploinsufficiency of TBK1 causes ALS and fronto-temporal dementia.

A randomized, placebo-controlled trial of memantine for functional disability in amyotrophic lateral sclerosis

Abstract Our objective is to describe the results of a phase II/III, 12-months, double-blinded, single-centre, randomized, parallel (1:1), clinical trial performed to evaluate the efficacy and safety of memantine in ALS. Patients with probable or definite ALS of less than 36 months disease duration and progression over a one-month lead-in period were randomly assigned to placebo or memantine at 20 mg/day. The primary endpoint was 12-months ALSFRS decline. Forced vital capacity, manual muscle testing, visual analogue scale, quality of life, motor unit number estimation and neurophysiological index were the secondary endpoints. The number of patients included was based on the assumption of a 50% change in the ALSFRS decline. Safety and adverse events were evaluated. Sixty-three patients were included in the trial. Memantine did not show more adverse events or laboratory changes than placebo. Primary and secondary outcomes were not different between groups by intention-to-treat and per-protocol analysis. The most sensitive measurements were neurophysiological, which declined linearly over time. In conclusion, the results of this study show that memantine is well tolerated and safe in ALS patients. We did not observe any evidence of efficacy for memantine but we cannot exclude a positive outcome on survival.

Home telemonitoring of non-invasive ventilation decreases healthcare utilisation in a prospective controlled trial of patients with amyotrophic lateral sclerosis

Background Non-invasive ventilation (NIV) is an efficient method for treating respiratory failure in patients with amyotrophic lateral sclerosis (ALS). However, it requires a process of adaptation not always achieved due to poor compliance. The role of telemonitoring of NIV is not yet established. Objectives To test the advantage of using modem communication in NIV of ALS patients. Design Prospective, single blinded controlled trial. Population and methods According to their residence, 40 consecutive ventilated ALS patients were assigned to one of two groups: a control group (G1, n=20) in which compliance and ventilator parameter settings were assessed during office visits; or an intervention group (G2, n=20) in which patients received a modem device connected to the ventilator. The number of office and emergency room visits and hospital admissions during the entire span of NIV use and the number of parameter setting changes to achieve full compliance were the primary outcome measurements. Results Demographic and clinical features were similar between the two groups at admission. No difference in compliance was found between the groups. The incidence of changes in parameter settings throughout the survival period with NIV was lower in G2 (p<0.0001) but it was increased during the initial period needed to achieve full compliance. The number of office or emergency room visits and inhospital admissions was significantly lower in G2 (p<0.0001). Survival showed a trend favouring G2 (p=0.13). Conclusions This study shows that telemonitoring reduces health care utilisation with probable favourable implications on costs, survival and functional status.

A blinded international study on the reliability of genetic testing for GGGGCC-repeat expansions in C9orf72 reveals marked differences in results among 14 laboratories

Background The GGGGCC-repeat expansion in C9orf72 is the most frequent mutation found in patients with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Most of the studies on C9orf72 have relied on repeat-primed PCR (RP-PCR) methods for detection of the expansions. To investigate the inherent limitations of this technique, we compared methods and results of 14 laboratories. Methods The 14 laboratories genotyped DNA from 78 individuals (diagnosed with ALS or FTD) in a blinded fashion. Eleven laboratories used a combination of amplicon-length analysis and RP-PCR, whereas three laboratories used RP-PCR alone; Southern blotting techniques were used as a reference. Results Using PCR-based techniques, 5 of the 14 laboratories got results in full accordance with the Southern blotting results. Only 50 of the 78 DNA samples got the same genotype result in all 14 laboratories. There was a high degree of false positive and false negative results, and at least one sample could not be genotyped at all in 9 of the 14 laboratories. The mean sensitivity of a combination of amplicon-length analysis and RP-PCR was 95.0% (73.9–100%), and the mean specificity was 98.0% (87.5–100%). Overall, a sensitivity and specificity of more than 95% was observed in only seven laboratories. Conclusions Because of the wide range seen in genotyping results, we recommend using a combination of amplicon-length analysis and RP-PCR as a minimum in a research setting. We propose that Southern blotting techniques should be the gold standard, and be made obligatory in a clinical diagnostic setting.

Paraspinal and limb motor neuron involvement within homologous spinal segments in ALS

OBJECTIVE We studied the involvement of motor neuron groups innervating paraspinal muscles in amyotrophic lateral sclerosis (ALS) and evaluated the value of paraspinal muscle EMG in the diagnosis of the disease. METHODS We used quantitative concentric needle EMG to study denervation and reinnervation in a paraspinal muscle and a limb muscle innervated by the C6 and L5 segments in 32 patients with ALS. As control subjects we studied 11 patients with peripheral neuropathy, and 46 non-neurogenic control subjects. RESULTS We found similar abnormalities in motor-unit potentials (MUPs) in paraspinal and limb muscles in these two segments in ALS. Fasciculation potentials (FPs) were more frequent in limb muscles than in paraspinal muscles and fibrillations and sharp waves (fibs-sw) were most frequent in tibialis anterior. In peripheral neuropathy paraspinal muscles were normal but tibialis anterior showed very abnormal motor unit potentials. CONCLUSIONS These results are consistent with generalised involvement of motor neurons in motor neuron pools in spinal segments in early stages of ALS progression. However, distally predominant fibrillations indicate susceptibility to ongoing denervation in reinnervated distal axons. Complex FPs of similar morphology to MUP analysis in the same early affected muscle suggests a proximal origin for these FPs at this phase. SIGNIFICANCE Our observations emphasize the value of paraspinal muscle EMG in the electrophysiological diagnosis of ALS.

Changes of the phrenic nerve motor response in amyotrophic lateral sclerosis: Longitudinal study

OBJECTIVE Phrenic nerve motor amplitude (Diaphr Ampl) is predictive of hypoventilation in amyotrophic lateral sclerosis (ALS). We aimed to evaluate its change over disease course and to correlate it to other measurements. METHODS Forty-nine unselected patients (35 men, 13 bulbar-onset, 56.5+/-8.9 years) with definitive or probable ALS were included. They were evaluated at entry (time 0) and 4-6 months (5.2+/-1.0) later (time 1) with: functional ALS rating scale (ALS-FRS) and respiratory subscore (ALS-FRSr); forced vital capacity (FVC); maximal inspiratory pressure (MIP); mean O(2) saturation overnight (SpO(2)mean); sniff maximal inspiratory pressure (SNIP); Diaphr Ampl and mean amplitude of the ulnar nerve response (ADM Ampl). RESULTS ALS-FRS, ALS-FRSr, Diaphr Ampl, FVC, SNIP, ADM Ampl (p<0.01) and SpO(2)mean (p<0.05) declined significantly. MIP did not change significantly (p=0.203). Coefficient of variation was similar for FVC, Diaphr Ampl, ADM Ampl and ALS-FRS but higher for SNIP. The percentage of change for Diaphr Ampl was significantly correlated to FVC and SNIP, but not to ADM Ampl or ALS-FRS. CONCLUSIONS Diaphr Ampl decreased significantly in a short period of time and its change is correlated to other respiratory tests. This test can be useful in patients with marked facial weakness or uncooperative. SIGNIFICANCE Diaphr Ampl is useful to monitor respiratory function in ALS patients and can be applied in clinical trials.

Percutaneous nocturnal oximetry in amyotrophic lateral sclerosis: Periodic desaturation

Percutaneous nocturnal oximetry (PNO) is useful to screen respiratory function in amyotrophic lateral sclerosis (ALS). PNO recordings of some patients disclose a periodical pattern of O2 desaturation (PP), whose significance is unknown. We aimed to characterize PP pattern, and we used a prospective study enrolling 261 consecutive ALS patients. Clinical, pulmonary and neurophysiological tests performed included: ALS functional rating scale, forced vital capacity (FVC), maximal inspiratory pressure (PImax), mouth occlusion pressure (MOP), phrenic nerve motor response, needle electromyography of the diaphragm, PNO, and sleep study. A total of 837 PNO recordings were analysed (3.2 recordings/patient) and 45 patients showed typical PP (17.2%). Four were excluded, 13 had normal diaphragm (group 1, G1), and in 28 the diaphragm was abnormal (G2). The two groups were comparable, apart from respiratory score, FVC and PImax which were lower in G2. In G1, REM sleep was absent and hypoventilation occurred at slow-wave sleep. Five patients in G1 were very spastic, had low MOP/FVC and a short survival. This study identified a subgroup of ALS patients (G1) with marked signs of upper motor neuron lesion, strong respiratory muscles, PP, low MOP/FVC ratio and poor prognosis. We speculate that they have a central respiratory dysfunction and deserve special care.

Phrenic nerve studies predict survival in amyotrophic lateral sclerosis

OBJECTIVE Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative disease associated with short survival due to respiratory failure. We aimed to test the predictive value of the phrenic nerve motor response for survival, in a large population of ALS patients. METHODS We included 254 ALS patients followed in our tertiary centre from 1997 to 2006, in whom phrenic nerve stimulation was performed according to the study inclusion and exclusion criteria. ALS was spinal onset in 175 and bulbar onset in 79 patients. The following features were recorded at entry: gender, age at presentation, onset region, diagnostic delay, forced vital capacity (FVC), ALS functional rating scale (ALS-FRS) including the respiratory subscore of the reviewed ALS-FRS and mean amplitude of motor responses by phrenic nerve stimulation (PhrenAmpl). RESULTS Survival analysis was evaluated by Kaplan-Meier log-rank test and multivariate Cox proportional hazards. Independent factors negatively affecting survival were bulbar onset, short diagnostic delay, FVC and small PhrenAmpl for the total population. Small PhrenAmpl and short diagnostic delay were also independent factors for both spinal and bulbar-onset patients; age at onset and FVC were also independent predictors in bulbar-onset patients. CONCLUSION Phrenic nerve stimulation is a non-volitional test that can be performed quickly in most patients; it is a powerful predictor of survival in ALS. SIGNIFICANCE Phrenic nerve stimulation should be considered as an additional test for respiratory assessment in ALS.

Association of paraspinal and diaphragm denervation in ALS

Paraspinal EMG needle examination is commonly performed in amyotrophic lateral sclerosis (ALS) for diagnosis. Because lower motor neurons for axial muscles and diaphragm are located medially in the anterior horn, we tested if involvement of axial muscles is associated with diaphragm weakness in ALS. Forty-four ALS patients were included with ALSFRS greater than 20/40. We used needle EMG to search for signs of denervation in biceps, tibialis anterior, C6 and T5 paraspinal muscles, and intercostal and diaphragm muscles. We also evaluated phrenic nerve motor responses and forced vital capacity (FVC). We tested specificity, sensitivity, and discriminative strength (ROC analysis). Fibs-sw in C6 and T5 paraspinal muscles, as well as fibs-sw in diaphragm and intercostal muscles showed high specificity and positive predictive value for FVC<80%. Discriminative strength was good for all the above tests, as well as for phrenic nerve amplitude and ALSFRS regarding FVC<80%. Axial muscles denervation is related to diaphragm denervation and therefore to poor respiratory function in ALS. We suggest that medially located lower motor neurons are affected concurrently in ALS.

What is the relevance of percutaneous endoscopic gastrostomy on the survival of patients with amyotrophic lateral sclerosis

Percutaneous endoscopic gastrostomy (PEG) is a standard procedure for feeding dysphagic amyotrophic lateral sclerosis (ALS) patients. Nevertheless, the effect of prognostic factors influencing survival after PEG remains unclear. We aimed to evaluate the prognostic value of several clinical features on survival after PEG placement. This study investigated 151 patients with ALS, in whom a PEG was inserted over the last 16 years in our centre. Survival curves were determined by Kaplan-Meier and the analysis of potential prognostic factors was performed by a Cox regression model. The overall median survival was 32 months, longer in spinal-onset disease patients − 42 vs. 29 months in bulbar-onset patients (p < 0.001). Median survival after PEG placement was 7.5 months, similar in both bulbar- and spinal-onset patients, 7.9 vs. 7.1 months, respectively. Thirteen percent of patients died within one month after PEG placement; this short-term survival was influenced by low forced vital capacity (FVC < 50%). In a multivariate analysis, only older age at disease onset was independently associated with poor outcome after PEG placement. In conclusion, survival after PEG placement was similar in bulbar- and spinal-onset patients, suggesting that the latter were in a more advanced stage at the time of PEG placement. Low FVC was associated with higher risk of short-term mortality. Older age at disease onset was associated with poorer outcome in bulbar-onset patients. Younger bulbar-onset patients are those who benefited most from PEG.