Annemarie Hübers

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.

Reply: Two novel mutations in conserved codons indicate that CHCHD10 is a gene associated with motor neuron disease

Sylvie Bannwarth, Samira Ait-El-Mkadem, Annabelle Chaussenot, Emmanuelle C. Genin, Sandra Lacas-Gervais, Konstantina Fragaki, Laetitia Berg-Alonso, Yusuke Kageyama, Valérie Serre, David Moore, Annie Verschueren, Cécile Rouzier, Isabelle Le Ber, Gaëlle Augé, Charlotte Cochaud, Françoise Lespinasse, Karine N’Guyen, Anne de Septenville, Alexis Brice, Patrick Yu-Wai-Man, Hiromi Sesaki, Jean Pouget and Véronique Paquis-Flucklinger

NEK1 mutations in familial amyotrophic lateral sclerosis

Sir, Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by adult-onset loss of motor neurons. Five to 10% of all ALS cases are familial ALS. To date, more than 20 genes have been implicated in causing familial ALS, with the discovery of mutations in CHCHD10 (Bannwarth et al. , 2014) and TBK1 (Cirulli et al. , 2015; Freischmidt et al. , 2015) representing the latest examples for monogenic causes of ALS. Most recently, whole exome sequencing of ALS patients suggested an association of heterozygous loss-of-function mutations in NEK1 with ALS. However, this observation was made in a cohort of mostly sporadic patients, and the result was only significant in a combined analysis of the discovery and the replication cohort (Cirulli et al. , 2015), making further validation essential. To assess the association between NEK1 variants and familial ALS we analysed whole exome sequence data of 265 familial ALS index patients and 827 control individuals. A subset of these exome sequence data has recently led to the discovery of mutations in TBK1 as a cause for ALS in an exome-wide mutational burden analysis (Freischmidt et al. , 2015). The patients with familial ALS were selected from families with two or more affected individuals from European countries (Germany, Sweden, Finland, Denmark, Switzerland, and Portugal) following a negative screen for SOD1 and C9orf72 mutations as described previously (Freischmidt et al. , 2015). In-house control exomes ( n = 827) from Germany were used to compare the variant burden in NEK1 . All ALS patients were diagnosed according to the EFNS Consensus criteria (Andersen et al. , 2012). Control subjects were comprised of healthy parents of children with various diseases, healthy control tissues of individuals with tumour diseases and 200 individuals of the KORA study. With informed written consent and approval by …

Polymerase chain reaction and Southern blot-based analysis of the C9orf72 hexanucleotide repeat in different motor neuron diseases

The GGGGCC-hexanucleotide repeat expansion in C9orf72 is the most common genetic cause of familial amyotrophic lateral sclerosis and frontotemporal dementia. This study determined the frequency of C9orf72 repeat expansions in different motor neuron diseases (amyotrophic lateral sclerosis (ALS), motor neuron diseases affecting primarily the first or the second motor neuron and hereditary spastic paraplegia). Whereas most studies on C9orf72 repeat expansions published so far rely on a polymerase chain reaction-based screening, we applied both polymerase chain reaction-based techniques and Southern blotting. Furthermore, we determined the sensitivity and specificity of Southern blotting of the C9orf72 hexanucleotide repeat in DNA derived from lymphoblastoid cell lines. C9orf72 repeat expansions were found in 27.1% out of 166 familial ALS patients, only once in 68 sporadic ALS patients, and not in 61 hereditary spastic paraplegia patients or 52 patients with motor neuron diseases affecting clinically primarily either the first or the second motor neuron. We found hints for a correlation between C9orf72 repeat length and the age of onset. Somatic instability of the C9orf72 repeat was observed in lymphoblastoid cell lines compared with DNA derived from whole blood from the same patient and therefore caution is warranted for repeat length determination in immortalized cell lines.

De novo FUS mutations are the most frequent genetic cause in early-onset German ALS patients.

In amyotrophic lateral sclerosis (ALS) patients with known genetic cause, mutations in chromosome 9 open reading frame 72 (C9orf72) and superoxide dismutase 1 (SOD1) account for most familial and late-onset sporadic cases, whereas mutations in fused in sarcoma (FUS) can be identified in just around 5% of familial and 1% of overall sporadic cases. There are only few reports on de novo FUS mutations in juvenile ALS patients. To date, no systematic evaluation on the frequency of de novo FUS mutations in early-onset ALS patients has been conducted. Here, we screened a cohort of 14 early-onset sporadic ALS patients (onset age <35 years) to determine the frequency of mutations in C9orf72, SOD1, and FUS in this defined patient cohort. All patients were recruited prospectively by a single center in a period of 38 months. No mutations were detected in SOD1 or C9orf72; however, we identified 6 individuals (43%) carrying a heterozygous FUS mutation including 1 mutation that has not been described earlier (c.1504delG [p.Asp502Thrfs*27]). Genetic testing of parents was possible in 5 families and revealed that the mutations in these patients arose de novo. Three of the 6 identified patients presented with initial bulbar symptoms. Our study identifies FUS mutations as the most frequent genetic cause in early-onset ALS. Genetic testing of FUS thus seems indicated in sporadic early-onset ALS patients especially if showing predominant bulbar symptoms and an aggressive disease course.

Status epilepticus: Clinical characteristics and EEG patterns associated with and without MRI diffusion restriction in 69 patients

OBJECTIVE Incidence and localization of cortical and thalamic peri-ictal diffusion-weighted imaging (DWI) abnormalities were investigated in patients with status epilepticus (SE). Clinical characteristics and EEG features were compared between patients with and without peri-ictal regional DWI restriction. Such correlations are important to improve the understanding of causes and significance of peri-ictal DWI restriction. METHODS We retrospectively investigated 69 SE-patients treated in our emergency department in whom SE was confirmed by EEG and MRI including DWI was performed. RESULTS 19/69 patients presented with peri-ictal DWI restriction: 18/19 with cortical restriction, 13 of them with additional thalamic restriction, and 1/19 with thalamic restriction only. 17/69 patients had DWI restrictions related to other pathologies, so that a possible overlap with peri-ictal DWI restriction could not be determined. In 33/69 patients no peri-ictal DWI restriction was detected. In contrast to SE-patients without DWI restriction, those with peri-ictal DWI restriction always presented with regional/unilateral epileptiform discharges (p<0.001). The EEG of SE-patients with peri-ictal DWI restriction was predominated by circumscribed periodic lateralized epileptiform discharges (PLEDs) (p<0.001) and by repetitive seizure patterns (p=0.009), while PLEDs occurred infrequently and EEG patterns were more variable in extent in patients without DWI restriction. Patients with peri-ictal diffusion had more often a quantitative disorder of consciousness (p=0.05) compared to patients without peri-ictal DWI restriction. No significant differences were found concerning age of patients, preceding generalized tonic-clonic seizures, and mortality. SIGNIFICANCE Patients with peri-ictal DWI restriction presented with a rather uniform EEG pattern characterized by circumscribed PLEDs possibly resulting from local cortical metabolic disturbances and with intermittent seizure patterns. The frequently observed quantitative disorder of consciousness despite circumscribed EEG patterns could be related to epileptic activity in the temporal lobe and cortico-thalamic synchronization. The higher percentage of bilateral status patterns and subcortical lesions in patients without peri-ictal DWI restrictions suggest a different pathomechanism.

Screening for CHCHD10 mutations in a large cohort of sporadic ALS patients: no evidence for pathogenicity of the p.P34S variant

Sir, Bannwarth et al. (2014) reported a mutation in CHCHD10 responsible for a variable phenotype including cerebellar ataxia, hearing impairment, myopathy, amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). This gene encodes the coiled-coil helix coiled-coil helix domain-containing protein 10 of largely unknown function. It is a strictly mitochondrially located protein that may be involved in respiratory chain function or mitochondrial genome stability (Bannwarth et al. , 2014). Several subsequent publications provided further evidence that CHCHD10 mutations can cause familial motor neuron disease/ALS (familial ALS), with an unusually slow disease progression in most patients (Bannwarth et al. , 2014; Johnson et al. , 2014; Muller et al. , 2014; Kurzwelly et al. , 2015; Penttila et al. , 2015). As an important argument for CHCHD10 causing ALS, co-segregation with disease could be demonstrated for the three CHCHD10 variants p.R15L (Johnson et al. , 2014; Muller et al. , 2014; Kurzwelly et al. , 2015), p.S59L (Bannwarth et al. , 2014) and p.G66V (Penttila et al. , 2015). The strongest genetic evidence for causality exists with regard to the p.G66V mutation, which was first detected by Muller et al. (2014) in a single patient with familial ALS and subsequently shown to co-segregate with a slowly progressing motor neuron disease in a total of 17 pedigrees (Penttila et al. , 2015). Hence there is meanwhile unequivocal evidence that CHCHD10 mutation can cause familial motor neuron degeneration. No association of CHCHD10 variants with ALS could be shown in genome-wide association studies, which can plausibly be explained by the low frequency of CHCHD10 mutations in familial ALS patient cohorts (Bannwarth et al. , 2014; Johnson et al. , 2014; Muller et al. , 2014). Consequently, due to the …

Hot-spot KIF5A mutations cause familial ALS

Brenner et al. show that mutations in a C-terminal hotspot of kinesin-5A (KIF5A) can cause a classical ALS phenotype. Experiments using patient-derived cell lines suggest haploinsufficiency as the molecular genetic mechanism. This underlines the relevance of intracellular transport processes for ALS, and is important for clinico-genetic diagnosis and counselling.

Sequence variations in C9orf72 downstream of the hexanucleotide repeat region and its effect on repeat-primed PCR interpretation: a large multinational screening study

Abstract A large GGGGCC-repeat expansion mutation (HREM) in C9orf72 is the most common known cause of ALS and FTD in European populations. Sequence variations immediately downstream of the HREM region have previously been observed and have been suggested to be one reason for difficulties in interpreting RP-PCR data. Our objective was to determine the properties of these sequence variations with regard to prevalence, the range of variation, and effect on disease prognosis. We screened a multi-national cohort (n = 6981) for the HREM and samples with deviant RP-PCR curves were identified. The deviant samples were subsequently sequenced to determine sequence alteration. Our results show that in the USA and European cohorts (n = 6508) 10.7% carried the HREM and 3% had a sequence variant, while no HREM or sequence variants were observed in the Japanese cohort (n = 473). Sequence variations were more common on HREM alleles; however, certain population specific variants were associated with a non-expanded allele.In conclusion, we identified 38 different sequence variants, most located within the first 50 bp downstream of the HREM region. Furthermore, the presence of an HREM was found to be coupled to a lower age of onset and a shorter disease survival, while sequence variation did not have any correlation with these parameters.

Pathological laughing and crying in amyotrophic lateral sclerosis is related to frontal cortex function

The syndrome of pathological laughing and crying (PLC) is characterized by episodes of involuntary outbursts of emotional expression. Although this phenomenon has been referred to for over a century, a clear-cut clinical definition is still lacking, and underlying pathophysiological mechanisms are not well understood. In particular, it remains ill-defined which kind of stimuli—contextually appropriate or inappropriate—elicit episodes of PLC, and if the phenomenon is a result of a lack of inhibition from the frontal cortex (“top-down-theory”) or due to an altered processing of sensory inputs at the brainstem level (“bottom-up-theory”). To address these questions, we studied ten amyotrophic lateral sclerosis (ALS) patients with PLC and ten controls matched for age, sex and education. Subjects were simultaneously exposed to either emotionally congruent or incongruent visual and auditory stimuli and were asked to rate pictures according to their emotional quality. Changes in physiological parameters (heart rate, galvanic skin response, activity of facial muscles) were recorded, and a standardized self-assessment lability score (CNS-LS) was determined. Patients were influenced in their rating behaviour in a negative direction by mood-incongruent music. Compared to controls, they were influenced by negative stimuli, i.e. they rated neutral pictures more negatively when listening to sad music. Patients rated significantly higher on the CNS-LS. In patients, changes of electromyographic activity of mimic muscles during different emotion-eliciting conditions were explained by frontal cortex dysfunction. We conclude that PLC is associated with altered emotional suggestibility and that it is preferentially elicited by mood-incongruent stimuli. In addition, physiological reactions as well as behavioural changes suggest that this phenomenon is primarily an expression of reduced inhibitory activity of the frontal cortex, since frontal dysfunction could explain changes in physiological parameters in the patient group. We consider these findings being important for the clinical interpretation of emotional reactions of ALS patients.