**6. Conclusion and future research**

The use of linkage analysis, candidate gene studies, and GWAS has led to the identification of several causal loci and genes for fALS and sALS. The overview above clearly shows the extent of heterogeneity in genes that underlie fALS, let alone sALS, illustrating the complex molecular basis of this disease. There is not one dominant biological process that is represented by these genes, although RNA-processing, axonal transport and synaptic dysfunction appear to emerge as being relevant in ALS etiology. Interestingly, several of the genes implicated in these processes are already known to be causal or have been implicated in other neurodegenerative diseases which suggests that there is, at least in part, a common underlying mechanism.

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This work has received funding from the European Community's Health Seventh Framework Programme (FP7/2007-2013) under grant agreement n° 259867, VSB fonds, EURO-MOTOR FP7, The Netherlands ALS Foundation (J.H.V, and L.H.v.d.B.), Neuroscience and Cognition Utrecht (NCU), the Prinses Beatrix Fonds (Kersten Foundation) and the Adessium Foundation (R.J.P., J.H.V. and L.H.v.d.B.). J.H.V. is supported by the Brain Foundation of The Netherlands and J.H.V, R.J.P., and M.K are supported by the

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**7. Acknowledgements** 

Thierry Latran Foundation.

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**8. References** 

Since these findings explain only about a third of the genetic variability in fALS and a small percentage of the genetic contribution to sALS, there is a clear need for the identification of additional causal loci. This would require the collection of large family pedigrees with many affected individuals, which is difficult in ALS considering the adult onset with rapid disease progression. However, the development of next generation sequencing techniques provides a possible solution to this problem. Using exome and whole-genome sequencing, causal genes can be identified with a small number of affected and unaffected individuals as has been shown in several, mostly autosomal recessive disorders (Choi et al., 2009; Ng et al., 2009). Recently, exome sequencing in two affected individuals from the same family identified *VCP* as a causal gene for fALS, illustrating that this technique is a promising tool for gene identification in ALS as well (Johnson et al., 2010). In addition, the repeat expansion in C9ORF72 was also discovered with the use of whole-genome sequencing (Renton et al., 2011).

The identification of causal genes for ALS has broadened our understanding of this motor neuron degenerative disease. Studying the function of associated genes in neurons and animal models has revealed several possible processes underlying ALS such as RNA processing, axonal transport, glutamate regulation, oxidative stress and synaptic dysfunction. However, the contribution of most genes to ALS pathogenesis has not been resolved. SOD1 and TDP-43 transgenic animal models have provided valuable insights into ALS pathogenesis. Further research using existing animal models of ALS associated genes and the generation of new animal models are needed to further determine their role in the disease. Generation of animal models harbouring repeat expansions in C9ORF72 and ATXN2 could help to reveal the pathogenic mechanisms behind these repeats. The effect of overexpression or knockdown of ALS associated genes and the expression of repeat expansions in motor neurons or motor neuron-like cell lines on protein aggregation and cell survival could also help to unravel the contribution of these genes to ALS. In addition, some associated genes (e.g. *DCTN1*, *PON1/2/3*, *TAF15*, and *VCP*) remain to be sequenced in larger cohorts from different populations in order to determine the actual contribution of these genes to ALS.

Additional new strategies in sALS include a more network oriented approach to gene identification. It is possible to detect networks of genes, proteins and metabolites that are misregulated in ALS, or that determine disease progression. By searching for subtle genetic variation that drives these network perturbations, new genes might be identified that are hard to detect with GWAS. Also, the focus in ALS genetics thus far has been on common variation in exonic DNA. The regulatory part of the genome is challenging to study, but might be relevant as well. This also requires the combined analysis of gene-expression and protein data with data on genetic variation. In addition, recent studies show that tandem repeats in DNA might be also relevant, as exemplified by the ATXN2 and C9ORF72 findings. Typically, this type of variation is hard to detect by current high-throughput methods. Lastly, the type of copy number variation that has not yet been covered very well to date, including variation in microRNAs or inversions, deserves more attention.

In summary, impressive progress in the understanding of the genetics of ALS has been made over the past several years with the identification of several causal genes. However, most of the genetic variability underlying ALS remains to be identified. The use of deep sequencing techniques and functional research will be needed to further broaden our understanding of ALS pathogenesis.
