Hanhui Fu

Excessive daytime sleepiness in Chinese patients with sporadic amyotrophic lateral sclerosis and its association with cognitive and behavioural impairments

Objective To examine the frequency and clinical features of excessive daytime sleepiness (EDS) and its association with cognitive and behavioural impairments in patients with amyotrophic lateral sclerosis (ALS). Methods We conducted a cross-sectional investigation to explore the frequency and clinical features of EDS in a group of 121 Chinese patients with ALS compared with 121 age-matched and sex-matched healthy subjects. EDS was diagnosed using the Epworth Sleepiness Scale (ESS). Other characteristics of patients with ALS including sleep quality, REM sleep behaviour disorder (RBD), restless legs syndrome (RLS), cognition, behaviour, depression and anxiety were also evaluated. Results EDS was significantly more frequent in patients with ALS than in controls (26.4% vs 8.3%; p<0.05). Patients with ALS with EDS scored lower scores on the revised ALS Functional Rating Scale (ALSFRS-R), Mini-Mental State Examination (MMSE), Montreal Cognitive Assessment (MoCA) and MMSE and MoCA delayed memory subitems and higher on the Frontal Behavioural Inventory (FBI) than patients with ALS without EDS. ESS scores correlated with global ALSFRS-R, FBI, MMSE and MoCA scores and MMSE and MoCA delayed memory scores. RLS and global ALSFRS-R scores were independently associated with EDS in patients with ALS. Conclusions We identified a high frequency of EDS symptoms in Chinese patients with ALS, and these patients might have more serious physical, cognitive and frontal behaviour impairment. Patients with ALS might improve quality of life from the timely recognition and optimised management of EDS symptoms. Our results further suggest that ALS is a heterogeneous disease that might exhibit abnormal sleep-wake patterns.

ANXA11 mutations prevail in Chinese ALS patients with and without cognitive dementia

Objective To investigate the genetic contribution of ANXA11, a gene associated with amyotrophic lateral sclerosis (ALS), in Chinese ALS patients with and without cognitive dementia. Methods Sequencing all the coding exons of ANXA11 and intron-exon boundaries in 18 familial amyotrophic lateral sclerosis (FALS), 353 unrelated sporadic amyotrophic lateral sclerosis (SALS), and 12 Chinese patients with ALS-frontotemporal lobar dementia (ALS-FTD). The transcripts in peripheral blood generated from a splicing mutation were examined by reverse transcriptase PCR. Results We identified 6 nonsynonymous heterozygous mutations (5 novel and 1 recurrent), 1 splice site mutation, and 1 deletion of 10 amino acids (not accounted in the mutant frequency) in 11 unrelated patients, accounting for a mutant frequency of 5.6% (1/18) in FALS, 2.3% (8/353) in SALS, and 8.3% (1/12) in ALS-FTD. The deletion of 10 amino acids was detected in 1 clinically undetermined male with an ALS family history who had atrophy in hand muscles and myotonic discharges revealed by EMG. The novel p. P36R mutation was identified in 1 FALS index, 1 patient with SALS, and 1 ALS-FTD. The splicing mutation (c.174-2A>G) caused in-frame skipping of the entire exon 6. The rest missense mutations including p.D40G, p.V128M, p.S229R, p.R302C and p.G491R were found in 6 unrelated patients with SALS. Conclusions The ANXA11 gene is one of the most frequently mutated genes in Chinese patients with SALS. A canonical splice site mutation leading to skipping of the entire exon 6 further supports the loss-of-function mechanism. In addition, the study findings further expand the ANXA11 phenotype, first highlighting its pathogenic role in ALS-FTD.

Genetic analysis of TIA1 gene in Chinese patients with amyotrophic lateral sclerosis

Mutations in the low-complexity domain (LCD) of T cell-restricted intracellular antigen-1 (TIA1) was recently identified to be associated with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) in non-Hispanic white populations. We sequenced the TIA1 exons 11-13 encoding LCD in a series of 588 Chinese ALS/ALS-FTD patients (Familial ALSxa0= 29; Sporadic ALSxa0= 546; ALS-FTDxa0= 13) and 500 neurologically normal control subjects. We found a novel heterozygous missense mutation (c.973A>G, p.N325D) in a sporadic ALS patient, which suggests that TIA1 LCD mutations are not common in Chinese ALS/ALS-FTD.

Mutation analysis of KIF5A in Chinese amyotrophic lateral sclerosis patients

Amyotrophic lateral sclerosis (ALS) is an age-related fatal neurodegenerative orphan disorder that is characterized by progressive injury of both the upper and lower motor neurons. Recently, loss-of-function mutations predominately disrupting the C-terminal amino acid sequence of KIF5A via aberrant exon 27 splicing have been reported in European ALS cohorts. However, the contributions of KIF5A mutations in Asian patients with ALS remain unclear. KIF5A sequences, including exons 26 and 27, were analyzed in a large Chinese ALS cohort comprising 33 unrelated familial ALS probands, 645 sporadic ALS (SALS) patients, 15 ALS patients presenting with concomitant frontotemporal dementia, 400 in-house controls, and 12,951 East Asian individuals from the Exome Aggregation Consortium and Genome Aggregation Database databases. As a result, the previously reported canonical splicing site mutation c.2993-1G>A was found in 1 SALS patient, while no mutations were detected in familial ALS case or ALS patients presenting with concomitant frontotemporal dementia. The frequency of KIF5A mutations accounts for 0.16% (1/645) of Chinese SALS patients, implying that it is an uncommon genetic determinant of ALS in Chinese patients.

Restless Legs Syndrome in Chinese Patients With Sporadic Amyotrophic Lateral Sclerosis

Objective: To evaluate the frequency and clinical features of restless legs syndrome (RLS) in a group of Chinese patients with amyotrophic lateral sclerosis (ALS). Methods: 109 Patients included in this study fulfilled the revised El Escorial diagnostic criteria for clinically definite, probable and lab-supported probable ALS, and a group of 109 control subjects was matched for age and sex to the ALS group. Disease severity was assessed by the revised ALS functional rating scale (ALSFRS-R). The diagnosis of RLS was made according to the criteria of the International RLS Study Group. Other characteristics including sleep quality, excessive daytime sleepiness (EDS), REM sleep behavior disorder (RBD), depression and anxiety were also evaluated in ALS patients. Results: RLS was significantly more frequent in ALS patients than in control subjects (14.6 vs. 0.9%; P < 0.05). Compared to those without RLS, ALS patients with RLS reported a higher frequency of anxiety and EDS. ALS patients with RLS showed more severe legs dysfunction. EDS and legs function scores of the ALSFRS-R were independent factors significantly associated with RLS in ALS patients. Conclusions: Our findings suggest that Chinese ALS patients exhibit a high frequency of RLS symptoms and that these patients may benefit from recognition of the condition and optimized management of its symptoms. Moreover, ALS patients might cause circadian rhythms disturbance and our study further supports that ALS is a heterogeneous disorder involving multiple systems; further studies are needed to confirm these preliminary findings.

Brain Structural and Perfusion Signature of Amyotrophic Lateral Sclerosis With Varying Levels of Cognitive Deficit

Objective To characterize the patterns of brain atrophy and perfusion as measured by arterial spin labeling (ASL)-MRI, in amyotrophic lateral sclerosis (ALS) patients with varying levels of cognitive deficit, including ALS with frontotemporal dementia (FTD). Methods A total of 55 ALS patients and 20 healthy controls (HCs) were included, and all participants underwent neuropsychological assessments and MRI scans. According to their cognitive performance, ALS patients were further subclassified into ALS with normal cognition (ALS-Cn, nu2009=u200927), ALS with cognitive impairment (ALS-Ci, nu2009=u200917), and ALS-FTD (nu2009=u200911). Voxel-based comparisons of gray matter (GM) changes and cerebral blood flow (CBF) were conducted among the subgroups. Results The whole-brain comparisons of GM changes and CBF among ALS-Ci, ALS-Cn, and HCs were not significantly different. However, the ALS-FTD patients demonstrated a similar pattern of GM loss and hypoperfusion with more significant alterations in the left frontal and temporal lobe compared with the HCs, ALS-Cn, and ALS-Ci patients. Decreased CBF was found in many of the same brain areas wherein structural alterations occurred, although isolated GM loss and hypoperfusion were also observed. In addition, for both GM and CBF abnormalities, a similar pattern of changes was found in the comparisons of ALS-FTD vs. ALS-Ci, ALS-FTD vs. ALS-Cn, and ALS-FTD vs. HCs, with the differences being most significant between ALS-FTD and HCs. Conclusion The cognitive status of ALS patients is associated with different patterns of GM changes and cerebral perfusion. ASL-MRI might be a useful tool with which to investigate the pathological burden of ALS and to disclose the early signature of possible cognitive impairment.

BS04. Characteristics of respiratory dysfunction in amyotrophic lateral sclerosis: A clinical study in 159 cases

Introduction Amyotrophic lateral sclerosis(ALS) is a neurodegenerative disorder involving both upper and lower motor neurons with an average survival time of 3–5u202fyears. Respiratory deterioration is the main cause of ALS and affects almost all ALS patients in different courses. Currently, it is still controversial of the characteristics of respiratory deterioration in ALS patients, including the effect of the type of disease onset, as well as the progression rates. Methods We performed a retrospective cohort study including 159 ALS patients at Peking Union Medical College Hospital from June 2014 to April 2017. Age at symptom onset, sex, time form onset to PFT, ALSFRS-R score, onset and area involved were recorded. All patients were followed in March 2016 and Jun 2017 separately. Depending on the pattern of onset, patents were subdivided into two groups. One included patients with bulbar-onset ALS and the other included those with limb-onset. Results The mean age at symptom onset of 159 cases included was 52.9u202f±u202f10.1u202fyears old; the male: female ratio was 1.24:1. Bulbar-onset of symptoms was seen in 17.0% patients, while 81.6% presented with limb-onset. The time from symptom onset to PFT was shorter in the bulbar-onset group (7.0 vs. 11.5u202fm, pu202f=u202f0.004), as well as the time from symptom onset to diagnosis (8.0 vs. 12.0u202fm, pu202f=u202f0.027). Significant differences were showed of the age at symptom onset (pu202f=u202f0.025), time form symptom onset to diagnosis (pu202f=u202f0.026) and ALSFRS-R scores (pu202f=u202f0.004) between the two groups. The PFT parameters revealed a main characteristic restrictive type of dysfunction. Only 26.0% patients of respiratory dysfunction from PFT parameters had respiratory complaints. Mean FVC% and FEV1%value was 86.7%u202f±u202f22.2%, 85.6%u202f±u202f19.2% respectively. After adjusting for sex, ALSFRS-R score, time from symptom onset to diagnosis, BMI and age at symptom onset, no significant difference was observed for patients with time form symptom onset to PFT less than 14 mouths. In contrast, for those with time more than 14 mouth, bulbar-onset group showed worse PFT values than the limb-onset group, with differences was 27.5% (95%CI: 4.3%, 50.7%), 24.7% (95%CI: 4.3%, 45.2%) and 42.0% (95%CI: 17.7%, 66.4%) separately. The decline rates for FVC% and FEV1% were 0.7% per mouth and 0.8% per mouth. No difference was found between the two groups. Δ FVC% and Δ FEV1% of patients in bulbar-involved group were 1.5% (95%CI: 0.0–18.5%) and 1.4% (95%CI: 0.0–9.8%), which were significantly higher than those in limb-involved group of 0.2% (95%CI: 0.0–10.4) and 0.5%(95%CI: 0.0–8.0%) (pu202f Conclusion The main type of pulmonary dysfunction was restrictive pattern. Bulbar involvement predicted poorer respiratory function in ALS patients compared with limb-only involvement, no matter whether the involvement was the onset symptom.The progression rate was higher in the bulbar involved patients than those with limb-only involvement.

F45. Genetic analysis of TIA1 gene in Chinese patients with amyotrophic lateral sclerosis

Introduction Amyotrophic lateral sclerosis (ALS) is a rapidly progressive lethal neurodegenerative disorder characterized by affecting the upper and lower motor neurons of the brain, brainstem and spinal cord. Approximately 15% of ALS patients also suffering from frontotemporal dementia (FTD). Very recently, six missense mutations affecting the low-complexity domain (LCD) of T cell-restricted intracellular antigen-1 (TIA1) were identified in 1039 non-Hispanic white ALS and ALS-FTD patients. Mutations in the TIA1 gene were suggest to account for ∼ 2% FALS and 0.5% sporadic ALS (SALS). Those data suggest that TIA1 mutations might be a rare cause of ALSu202f±u202fFTD. Studies analyzing Asian ALS patient cohorts have not been reported. Previous studies have shown that mutations in the ALS causal genes varied among ethnic populations, such as the G4C2 repeat expansion in the C9orf72 gene is rare in Asian people. To investigate the frequency and spectrum of TIA1 mutations further, we firstly performed mutational screening of TIA1 in a large cohort of ALS/ALS-FTD patients of Chinese origin. Methods The study included 576 Chinese ALS/ALS-FTD patients (FALSu202f=u202f28; SALSu202f=u202f535; ALS-FTDu202f=u202f13) and 500 neurologically normal control subjects. All participants were of Han descent and from mainland China. ALS/ALS-FTD patients were registered at ALS clinic of Neurology Department, Peking Union Medical College Hospital from January 2016 to August 2017. TIA1 (NM_022173) exons 11, 12 and 13 encoding the prion-like LCD domain of TIA1 were sequenced in all 576 cases. Resulting chromatograms were aligned to reference human genome ( UCSC hg19) using CodonCode Aligner tool. All patients harboring mutations of the TIA1 were examined other common ALS causal genes including SOD1, ANXA11, FUS, OPTN, UBQLN2, TARDB and C9orf72. Besides, the identified mutations were subsequently screened in 500 controls. Results TIA1 sequence analysis revealed a novel heterozygous missense mutation, c.973A > G (p.N325D), in a sporadic ALS case. The variant was not present in our 500 neurologically normal controls as well as several public data bases includingExAC, gnomAD, 1000 Genomes Project and SNP150. The p.N325D substitution affects an highly conserved amino acid in the prion-like LCD domain. Moreover, Four distinct programs – PolyPhen2, Mutation Taster, SIFT and CADD – all predicted p.N325D as a damaging mutation. Conclusion In conclusion, we have identified a novel mutation in the TIA1 gene in SALS patients of Chinese origin. Our findings provide an overview of the TIA1 mutation in Chinese ALS patients. We propose that the TIA1 mutation is not a frequent mutation in Chinese ALS patients.

S55. ANXA11 mutations prevail in Chinese ALS patients with and without cognitive dementia

Introduction Amyotrophic lateral sclerosis (ALS) is a fatal neurological disease characterized by progressive paralysis and ultimately respiratory failure within 5u202fyears of symptom onset. Approximately 5–10% of ALS cases exhibit familial inheritance (FALS) and causative gene mutations can be found in 60% of FALS patients. The remaining 90–95% of ALS cases show sporadic manner (SALS) and mutations in the same genes are responsible for 10% of SALS patients. To date, rare variants in more than 30 genes have been reported to cause or be associated with ALS. Recently, Smith and et al. identified mutations in ANXA11 in ALS patients of European ancestry but pathogenicity of ANXA11 in other ALS cohorts remained unproved. In the current study, we investigated ANXA11 mutations in Chinese ALS patients with or without cognitive decline. Methods: Study population A total of 383 Chinese ALS patients were recruited at ALS clinic of Neurology Department, Peking Union Medical College Hospital (PUMCH) from January 2016 to August 2017. Mutation screening All coding exons and at least 100u202fbp of flanking introns of ANXA11(NM_145869) were sequenced using ABI 3730 automated DNA-sequencing system. Rare variants (MAFu202f Transcripts investigation of the splicing mutation Total RNA was extracted using TRIzol (Invitrogen) from peripheral white blood cells. Then 2.5u202f μ g RNA were used to perform Reverse transcriptase-PCR. cDNA was amplified using primer pair ANXA11-RT-F(5′-CCATGAGCTACCCTGGCTAT-3′) and ANXA11-RT-R(5′-GACTCCCCAGGCAGTCAAT-3′) locating at ANXA11 exon 4 and exon 8 (shown in Fig. 3), respectively. The PCR products were isolated on a 1.5% agarose gel. DNA fragments of interest were gel-purified and sequenced. Results We identified 6 nonsynonymous heterozygous mutations (c.107C>G, p.P36R; c.119A>G, p.D40G; c.382G>A, p.V128M; c.687T>A, p.S229R; c.904C>T, p.R302C; c.1471G>A, p.G491R) and 1 splice-site mutation(c.174-2A>G, p.A58_Q187del) in 10 unrelated patients, accounting for a mutant frequency of 5.6% (1/18) in FALS, 2.3% (8/353) in SALS and 8.3% (1/12) in ALS-FTD. A novel p. P36R mutation was identified in one FALS index, one SALS patient and one ALS-FTD. The splicing mutation(c.174-2A>G) caused in-frame skipping of the entire exon 6 and further supported the loss-of-function mechanism. Conclusion ANXA11 gene is the most frequently mutated genes in Chinese patients with SALS. The study findings further expand the ANXA11 phenotype, firstly highlighting its pathogenic role in ALS-FTD.

F47. Screening of GLE1 mutations in Chinese amyotrophic lateral sclerosis patients

Introduction Amyotrophic lateral sclerosis (ALS) is a fatal disease involving both upper and lower motor neuron, resulting in a 3–5u202fyear of survival from symptom onset caused by rapidly progressive paralysis and respiratory failure. It is etiologically heterogeneous. Approximately 90% of ALS cases are sporadic (SALS) with unknown causes. But The remaining 10% of patients have a family history (FALS) and harbor a causative gene mutation, reflecting genetic factor play an important role in the pathogenesis of ALS. To date, mutations in over 20 genes have been reported in ALS populations. Mutations in GLE1 are the causation of two fetal motoneuron diseases: lethal congenital contracture syndrome 1 (LCCS1) and lethal arthrogryposis with anterior horn cell disease (LAAHD). Recently, Kaneb et al. identified GLE1 variants within sporadic and familial Caucasian ALS cases, supporting defects in RNA metabolism were associated with pathogenesis of ALS. However, GLE1 mutations have not been investigated in Chinese ALS patients so far. Accordingly, we evaluated whether GLE1 mutations harbored in ALS patients from mainland China. Methods The current study recruited probands of 20 ALS families (mean age of onset was 50.1u202f±u202f10.9u202fyears; 10 females, 10 males), 230 SALS patients (mean age of onset was 52.1u202f±u202f11.9u202fyears; 96 females, 134 males). All 16 exons (plus 50u202fbp of flanking introns) of GLE1 (NM_001003722.1) including the entire coding region were amplified using polymerase chain reaction (PCR). The reaction conditions of PCR were as follows: 5u202fmin of denaturation at 95u202f°C, 8 cycles were used with 20u202fs at 94u202f°C, 20u202fs at 65u202f°C (decreasing by 1u202f°C with each cycle) and 30u202fs at 72u202f°C. 20u202fs at 94u202f°C, 20u202fs at 58u202f°C and 30u202fs at 72u202f°C for the subsequent 34 cycles, a final extension step at 72u202f°C for 5u202fmin. PCR products were subjected to Sanger sequencing using ABI 3730 automated DNA-sequencing system (Applied Biosystems). The sequences were aligned with reference sequence using CodonCode Aligner. Results GLE1 mutation screening of 250 ALS cases revealed 4 synonymous genetic variants(c.444G > A, p. R148R; c.946C > A, p. R316R; c.1641T > C, p. Y547Y; c.2082C > T, P. S694S), which presented in dbSNP database. Bioinformatic analysis using ESE finder and Human Splicing Finder 3.0 showed that all the silent mutations did not affect transcription process. We did not identify any novel or reported rare variants in all 16 coding exons of GLE1 from 250 Chinese ALS patients. Conclusion In summary, our results suggested that GLE1 mutations are not common in FALS and SALS of Chinese origin. Further study in larger and additional populations would be required to evaluate the role of GLE1 in ALS causation.