**4. ALS-FTD2 (9p13.2-21.3)**

Several linkage studies associated **chromosome 9p** to ALS-FTD, designated as ALS-FTD2 (Table 3). The first two independent studies found linkage to locus 9p13.2-21.3 in a Dutch and a Scandinavian family (Morita et al., 2006; Vance et al., 2006). Subsequently, eight other linkage studies in families from Canada, France, Belgium, North-America, Australia and Wales showed association to regions on chromosome 9p13.1-q21 (Boxer et al., 2010; Gijselinck et al., 2010; Le Ber et al., 2009; Luty et al., 2009; Momeni et al., 2006; Pearson et al., 2011; Valdmanis et al., 2007; Yan et al., 2006). Individuals in these families were diagnosed with ALS, FTD, and ALS-FTD. However, dementia, psychosis and Parkinsonism were also seen. Besides the co-occurrence of ALS and FTD in families, there is also considerable clinical overlap between ALS and FTD, i.e. mild cognitive abnormalities occur in up to 50% of ALS patients and in approximately 5% of ALS patients FTD is present with marked behavioral changes and language impairment (Elamin et al., 2011; Ringholz et al., 2005). Furthermore, ALS and FTD are both characterized by TDP-43 positive ubiquitinated cytoplasmic inclusions (Neumann et al., 2006). This strongly supports the idea that there is a common genetic contribution to the pathogenesis of both diseases.

A total of 41 genes, four micro RNAs, two pseudogenes, and a non-coding RNA in the associated chromosome 9p region have been screened for mutations but only in one study a premature stop codon in the intraflagellar transport 74 gene (*IFT-74*) was identified in two brothers from one family (Momeni et al., 2006). However, no mutations in *IFT-74* were identified in any of the other ALS-FTD families that were linked to chromosome 9p and it is therefore unlikely that this mutation is the underlying cause in these families.

Interestingly, a recent GWAS in sALS patients found association between ALS and chromosome 9p21.2. 2323 ALS patients and 9013 controls were genotyped and genomewide significance was found for SNPs on these two loci (van Es et al., 2009c). This finding was replicated in a second, independent cohort of 2532 ALS patients and 5940 controls. The associated SNPs are in a 80-kb linkage disequilibrium (LD) block on chromosome 9 which overlaps with the common region found in the ALS-FTD linkage studies (Figure 1).

decreased KIFAP3 expression (Landers et al., 2009). However, two subsequent studies in two Italian cohorts could not replicate the finding that the CC genotype had a beneficial effect on survival or decreased KIFAP3 expression in ALS patients (Orsetti et al., 2011; Traynor et al., 2010). KIFAP3 is part of the trimeric kinesin 2 motor complex KIF3 which mediates binding between proteins and their cargo. It serves multiple functions including a role in mitosis and intracellular transport of organelles and proteins in various tissues

The largest GWAS to date identified two loci, on **chromosome 9p21.2** and **19p13.11**, to be associated with sALS. The genetic variant in 19p13.11 maps to a haplotype within the boundaries of the *UNC13A* gene. Two studies failed to replicate this finding, but were underpowered, and more studies are needed to firmly establish genetic variation in

The association to chromosome 9p21.2 will be discussed in more detail in the following

Several linkage studies associated **chromosome 9p** to ALS-FTD, designated as ALS-FTD2 (Table 3). The first two independent studies found linkage to locus 9p13.2-21.3 in a Dutch and a Scandinavian family (Morita et al., 2006; Vance et al., 2006). Subsequently, eight other linkage studies in families from Canada, France, Belgium, North-America, Australia and Wales showed association to regions on chromosome 9p13.1-q21 (Boxer et al., 2010; Gijselinck et al., 2010; Le Ber et al., 2009; Luty et al., 2009; Momeni et al., 2006; Pearson et al., 2011; Valdmanis et al., 2007; Yan et al., 2006). Individuals in these families were diagnosed with ALS, FTD, and ALS-FTD. However, dementia, psychosis and Parkinsonism were also seen. Besides the co-occurrence of ALS and FTD in families, there is also considerable clinical overlap between ALS and FTD, i.e. mild cognitive abnormalities occur in up to 50% of ALS patients and in approximately 5% of ALS patients FTD is present with marked behavioral changes and language impairment (Elamin et al., 2011; Ringholz et al., 2005). Furthermore, ALS and FTD are both characterized by TDP-43 positive ubiquitinated cytoplasmic inclusions (Neumann et al., 2006). This strongly supports the idea that there is a common genetic contribution to the

A total of 41 genes, four micro RNAs, two pseudogenes, and a non-coding RNA in the associated chromosome 9p region have been screened for mutations but only in one study a premature stop codon in the intraflagellar transport 74 gene (*IFT-74*) was identified in two brothers from one family (Momeni et al., 2006). However, no mutations in *IFT-74* were identified in any of the other ALS-FTD families that were linked to chromosome 9p and it is therefore unlikely that this mutation is the underlying cause in

Interestingly, a recent GWAS in sALS patients found association between ALS and chromosome 9p21.2. 2323 ALS patients and 9013 controls were genotyped and genomewide significance was found for SNPs on these two loci (van Es et al., 2009c). This finding was replicated in a second, independent cohort of 2532 ALS patients and 5940 controls. The associated SNPs are in a 80-kb linkage disequilibrium (LD) block on chromosome 9 which overlaps with the common region found in the ALS-FTD linkage

including neurons (Haraguchi et al., 2006; Takeda et al., 2000).

*UNC13A* as being causative to sALS.

**4. ALS-FTD2 (9p13.2-21.3)** 

pathogenesis of both diseases.

these families.

studies (Figure 1).

sections.


Table 3. Overview of linkage studies in ALS-FTD families. N.K. = not known

Since this initial report several other GWAS in ALS patients have replicated the association to chromosome 9p21.2. In a GWAS performed on 405 Finnish ALS patients, of whom 93 patients had fALS, and 497 control individuals two association peaks were identified (Laaksovirta et al., 2010). One peak corresponded to the autosomal recessive D90A allele of the SOD1 gene. The other was identified in a 232-kb LD block on chromosome 9p21.2. The association signals in this study were mainly driven by the 93 fALS patients. A 42-SNP risk haplotype across the chromosome 9p21 locus was shared between 41 fALS cases with an odds ratio of 21.0 (Laaksovirta et al., 2010). In another GWAS in an ALS cohort from the UK

Genetics of Amyotrophic Lateral Sclerosis 497

Further studies will be needed to characterize the C9ORF72 protein and to establish the

UNC13a is a member of UNC13 family of presynaptic proteins. The protein consists of 1791 amino acids but several isoforms exist. It contains a zinc-finger like C1 domain that is homologous to a diacylglycerol and phorbol ester binding region of protein kinase C (PKC), three C2 domains that are similar to the calcium binding regulatory regions of PKC and synaptotagmin, a calmodulin binding domain and two Munc homology domains (Basu et

In mammals, the Munc13 family comprises four homologous members, Munc13-1, Munc13-2, Munc13-3, and Munc13-4. Deletion mutants of Munc13-1 in mice, the murine homologue of UNC13a, shows that the protein is needed for presynaptic vesicle maturation and fusion competence in glutamergic hippocampal neurons (Augustin et al., 1999). GABA-ergic neurons in the hippocampus show no spontaneous or evoked synaptic transmission in absence of both Munc13-1 and Munc13-2 (Varoqueaux et al., 2002). Neuromuscular junction (NMJ) axon terminals contain Munc13-1 and a splice variant of Munc13-2 (Varoqueaux et al., 2005). Mice deficient in Munc13 due to a double knockout of Munc13-1 and Munc13-2 form specialized neuromuscular endplates. However, the distribution, size and shape of these synapses are altered. Also, muscle morphology is abnormal and a larger number of motor neurons is present in the spinal cord in Munc13- 1/2 knockout mice, probably as a result of defective apoptosis. Furthermore, evoked synaptic transmission is impaired in these mutants but spontaneous transmission is unchanged (Varoqueaux et al., 2005). This indicates that vesicle priming in NMJs is partially independent of Munc13-1 or Munc13-2. However, despite the unchanged spontaneous transmission, muscle innervation is aberrant in Munc13-1/2 knockout mice

As exemplified by the defects observed in Munc13-1 and Munc13-1/2 knockout mice, it is plausible that a disruption in UNC13a expression affects motor neurons and muscle innervation. The effect of UNC13a on glutamate exocytosis is also interesting since Riluzole, the only drug with a proven effect on ALS, is a glutamate release inhibitor. Therefore, *UNC13a* is an interesting candidate gene to be investigated further for a role in ALS

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

consequences of the intronic repeat on ALS pathogenesis.

**5.2 UNCoordinated 13 homolog A (UNC13a)** 

al., 2005).

(Varoqueaux et al., 2005).

underlying mechanism.

**6. Conclusion and future research** 

pathogenesis.

consisting of 599 ALS patients and 4144 control individuals two SNPs on chromosome 9p21.2 were found to be associated with ALS (Shatunov et al., 2010). A joint analysis including 4132 ALS patients and 8425 controls from this UK cohort and from previously published data from the UK, USA, Netherlands, Ireland, Italy, France, Sweden, and Belgium also showed significant association to the locus on chromosome 9p21.2 (Shatunov et al., 2010). In addition, replication of one of the associated SNPs on chromosome 9p21.2 was found in a GWAS performed in FTD patients when analyzing the ALS-FTD patients only. A different SNP in this locus was significantly associated with FTD (Rollinson et al., 2011). A trend towards significant genome-wide association between chromosome 9p21.2 and FTD was found when analyzing 426 FTD patients with TDP-43 pathology without mutations in the progranulin gene and 2509 control individuals (Van Deerlin et al., 2009). A replication study in Chinese and Japanese sALS patients failed to find association to one of the previously associated SNPs on chromosome 9p21.2 but this might be due to a lack of power (Iida et al., 2011).

In summary, linkage studies in ALS-FTD families and GWAS in sALS, ALS-FTD and FTD patients provide compelling evidence for a role of chromosome 9p21.2 in ALS and/or FTD.

As mentioned, recently two studies identified a GGGGCC hexanucleotide repeat expansion in intron 1 of the *C9ORF72* gene as the cause of chromosome 9p-linked ALS-FTD (Dejesus-Hernandez et al., 2011; Renton et al., 2011).
