The role of de novo mutations in the development of amyotrophic lateral sclerosis.

The genetic basis combined with the sporadic occurrence of amyotrophic lateral sclerosis (ALS) suggests a role of de novo mutations in disease pathogenesis. Previous studies provided some evidence for this hypothesis; however, results were conflicting: no genes with recurrent occurring de novo mutations were identified and different pathways were postulated. In this study, we analyzed whole-exome data from 82 new patient-parents trios and combined it with the datasets of all previously published ALS trios (173 trios in total). The per patient de novo rate was not higher than expected based on the general population (P = 0.40). We showed that these mutations are not part of the previously postulated pathways, and gene-gene interaction analysis found no enrichment of interacting genes in this group (P = 0.57). Also, we were able to show that the de novo mutations in ALS patients are located in genes already prone for de novo mutations (P < 1 × 10-15 ). Although the individual effect of rare de novo mutations in specific genes could not be assessed, our results indicate that, in contrast to previous hypothesis, de novo mutations in general do not impose a major burden on ALS risk.

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.

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.

Can lesions to the motor cortex induce amyotrophic lateral sclerosis?

A recent staging effort for amyotrophic lateral sclerosis (ALS) has demonstrated that the TDP-43 neuropathology may initiate focally in the motor cortex in the majority of patients. We searched our data bank for patients with lesions of the motor cortex which preceded disease onset. We performed a search of our patient- and MRI-data bank and screened 1,835 patients with amyotrophic lateral sclerosis for frontal lobe/motor cortex lesions. We found 18 patients with definite ALS who had documented and defined lesions of the motor cortex, which preceded the initial ALS symptoms by 8-42 years. In the vast majority (15/18) of the patients, the onset of ALS was closely related to the focal lesion since it started in a body region reflecting the damaged cortical area. The findings suggest that initial lesions to the motor cortex may be a contributing initiating factor in some patients with ALS or determine the site of onset in individuals pre-disposed to ALS.

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.