To identify novel genes associated with ALS, we undertook two lines of investigation. We carried out a genome-wide association study comparing 20,806 ALS cases and 59,804 controls. Independently, we performed a rare variant burden analysis comparing 1,138 index familial ALS cases and 19,494 controls. Through both approaches, we identified kinesin family member 5A (KIF5A) as a novel gene associated with ALS. Interestingly, mutations predominantly in the N-terminal motor domain of KIF5A are causative for two neurodegenerative diseases: hereditary spastic paraplegia (SPG10) and Charcot-Marie-Tooth type 2 (CMT2). In contrast, ALS-associated mutations are primarily located at the C-terminal cargo-binding tail domain and patients harboring loss-of-function mutations displayed an extended survival relative to typical ALS cases. Taken together, these results broaden the phenotype spectrum resulting from mutations in KIF5A and strengthen the role of cytoskeletal defects in the pathogenesis of ALS.

SUMMARY Exome sequencing is an effective strategy for identifying human disease genes. However, this methodology is difficult in late-onset diseases where limited availability of DNA from informative family members prohibits comprehensive segregation analysis. To overcome this limitation, we performed an exome-wide rare variant burden analysis of 363 index cases with familial ALS (FALS). The results revealed an excess of patient variants within TUBA4A, the gene encoding the Tubulin, Alpha 4A protein. Analysis of a further 272 FALS cases and 5,510 internal controls confirmed the overrepresentation as statistically significant and replicable. Functional analyses revealed that TUBA4A mutants destabilize the microtubule network, diminishing its repolymerization capability. These results further emphasize the role of cytoskeletal defects in ALS and demonstrate the power of gene-based rare variant analyses in situations where causal genes cannot be identified through traditional segregation analysis.

To identify novel genes associated with ALS, we undertook two lines of investigation. We carried out a genome-wide association study comparing 20,806 ALS cases and 59,804 controls. Independently, we performed a rare variant burden analysis comparing 1,138 index familial ALS cases and 19,494 controls. Through both approaches, we identified kinesin family member 5A (KIF5A) as a novel gene associated with ALS. Interestingly, mutations predominantly in the N-terminal motor domain of KIF5A are causative for two neurodegenerative diseases, hereditary spastic paraplegia (SPG10) and Charcot-Marie-Tooth Type 2 (CMT2). In contrast, ALS associated mutations are primarily located at the C-terminal cargo-binding tail domain and patients harboring loss of function mutations displayed an extended survival relative to typical ALS cases. Taken together, these results broaden the phenotype spectrum resulting from mutations in KIF5A and strengthen the role of cytoskeletal defects in the pathogenesis of ALS.

To identify genetic factors contributing to amyotrophic lateral sclerosis (ALS), we conducted whole-exome analyses of 1,022 index familial ALS (FALS) cases and 7,315 controls. In a new screening strategy, we performed gene-burden analyses trained with established ALS genes and identified a significant association between loss-of-function (LOF) NEK1 variants and FALS risk. Independently, autozygosity mapping for an isolated community in the Netherlands identified a NEK1 p.Arg261His variant as a candidate risk factor. Replication analyses of sporadic ALS (SALS) cases and independent control cohorts confirmed significant disease association for both p.Arg261His (10,589 samples analyzed) and NEK1 LOF variants (3,362 samples analyzed). In total, we observed NEK1 risk variants in nearly 3% of ALS cases. NEK1 has been linked to several cellular functions, including cilia formation, DNA-damage response, microtubule stability, neuronal morphology and axonal polarity. Our results provide new and important insights into ALS etiopathogenesis and genetic etiology.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are overlapping, fatal neurodegenerative disorders in which the molecular and pathogenic basis remains poorly understood. Ubiquitinated protein aggregates, of which TDP-43 is a major component, are a characteristic pathological feature of most ALS and FTD patients. Here we use genome-wide linkage analysis in a large ALS/FTD kindred to identify a novel disease locus on chromosome 16p13.3. Whole-exome sequencing identified a CCNF missense mutation at this locus. Interrogation of international cohorts identified additional novel CCNF variants in familial and sporadic ALS and FTD. Enrichment of rare protein-altering CCNF variants was evident in a large sporadic ALS replication cohort. CCNF encodes cyclin F, a component of an E3 ubiquitin–protein ligase complex (SCFCyclin F). Expression of mutant CCNF in neuronal cells caused abnormal ubiquitination and accumulation of ubiquitinated proteins, including TDP-43 and a SCFCyclin F substrate. This implicates common mechanisms, linked to protein homeostasis, underlying neuronal degeneration.

Amyotrophic lateral sclerosis (ALS) is a lethal motor neuron disease that progressively debilitates neuronal cells that control voluntary muscle activity. Biomarkers are urgently needed to facilitate ALS diagnosis and prognosis, and as indicators of therapeutic response in clinical trials. microRNAs (miRNAs), small posttranscriptional modifiers of gene expression, are frequently altered in disease conditions. Besides their important regulatory role in variety of biological processes, miRNAs can also be released into the circulation by pathologically affected tissues and display remarkable stability in body fluids. In a mouse model of ALS that expresses mutated human superoxide dismutase 1 (SOD1-G93A) skeletal muscle is one of the tissues affected early by mutant SOD1 toxicity. To find biomarkers for ALS, we studied miRNA alterations from skeletal muscle and plasma of SOD1-G93A mice, and subsequently tested the levels of the affected miRNAs in the serum from human ALS patients. Fast-twitch and slow-twitch muscles from symptomatic SOD1-G93A mice (age 90 days) and their control littermates were first studied using miRNA microarrays and then evaluated with quantitative PCR from five age groups from neonatal to the terminal disease stage (10–120 days). Among those miRNA changed in various age/gender/muscle groups (miR-206, -1, -133a, -133b, -145, -21, -24), miR-206 was the only one consistently altered during the course of the disease pathology. In both sexes, mature miR-206 was increased in fast-twitch muscles preferably affected in the SOD1-G93A model, with highest expression towards the most severely affected animals. Importantly, miR-206 was also increased in the circulation of symptomatic animals and in a group of 12 definite ALS patients tested. We conclude that miR-206 is elevated in the circulation of symptomatic SOD1-G93A mice and possibly in human ALS patients. Although larger scale studies on ALS patients are warranted, miR-206 is a promising candidate biomarker for this motor neuron disease.

Hexanucleotide repeat expansions within the C9orf72 gene are the most important genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The difficulty of developing a precise method to determine the expansion size has hampered the study of possible correlations between the hexanucleotide repeat number and clinical phenotype. Here we characterize, through a new non-radioactive Southern blot protocol, the expansion size range in a series of 38 ALS and 22 FTD heterozygous carriers of >30 copies of the repeat. Maximum, median and modal hexanucleotide repeat number were higher in ALS patients than in FTD patients (P< 0.05 in all comparisons). A higher median number of repeats correlated with a bigger range of repeat sizes (Spearman's ρ = 0.743, P = 1.05 × 10(-11)). We did not find any correlation between age of onset or disease duration with the repeat size in neither ALS nor FTD mutation carriers. Clinical presentation (bulbar or spinal) in ALS patients did not correlate either with the repeat length. We finally analyzed two families with affected and unaffected repeat expansion carriers, compared the size of the repeat expansion between two monozygotic (MZ) twins (one affected of ALS and the other unaffected), and examined the expansion size in two different tissues (cerebellum and peripheral blood) belonging to the same FTD patient. The results suggested that the length of the C9orf72 repeat varies between family members, including MZ twins, and among different tissues from the same individual.

A hexanucleotide repeat expansion in chromosome 9 open reading frame 72 (C9orf72) can cause amyotrophic lateral sclerosis (ALS) and/or frontotemporal dementia (FTD). We assessed its frequency in 781 sporadic ALS (sALS) and 155 familial ALS (fALS) cases, and in 248 Spanish controls. We tested the presence of the reported founder haplotype among mutation carriers and in 171 Ceph Europeans from Utah (CEU), 170 Yoruba Africans, 81 Han Chinese, and 85 Japanese subjects. The C9orf72 expansion was present in 27.1% of fALS and 3.2% of sALS. Mutation carriers showed lower age at onset (P = 0.04), shorter survival (P = 0.02), greater co‐occurrence of FTD (P = 8.2 × 10−5), and more family history of ALS (P = 1.4 × 10−20), than noncarriers. No association between alleles within the normal range and the risk of ALS was found (P = 0.12). All 61 of the mutation carriers were tested and a patient carrying 28 hexanucleotide repeats presented with the founder haplotype. This haplotype was found in 5.6% Yoruba Africans, 8.9% CEU, 3.9% Japanese, and 1.6% Han Chinese chromosomes.