**3.7 Peripherin (PRPH)**

Peripherin is an intermediate filament similar to neurofilaments and is also associated with axonal spheroids in the proximal axon of spinal cord motor neurons of ALS patients (Corbo and Hays, 1992). It is also present in Lewy body-like inclusions and Bunina bodies that are seen in a portion of ALS patients (He and Hays, 2004; Mizuno et al., 2011). Peripherin is predominantly expressed in the peripheral nervous system and in spinal motor neurons in the central nervous system. After neuronal injury, peripherin expression is upregulated in spinal motor neurons and this upregulation has been linked to axonal regeneration (Troy et al., 1990). However, transgenic mice with wild-type overexpression of peripherin develop a late-onset and selective motor neuron disease characterized by intermediate filament inclusions (Beaulieu et al., 1999). For these reasons, the possibility of *PRPH* mutations in

fALS case could not be tested and none of the missense mutations were predicted to be deleterious (Daoud et al., 2011). One study did not identify ALS specific variation in the

The paraoxonase gene cluster consists of 3 genes (*PON1, PON2,* and *PON3*) and is located in an 80-kb block on chromosome 7q21.3-22.1. PON1 and PON3 are primarily expressed in liver where they are associated with high-density lipoproteins, whereas PON2 is ubiquitously expressed (Costa et al., 2005; Draganov et al., 2000; Ng et al., 2002). Both PON1 and PON2 expression has been shown in mouse brain (Giordano et al., 2011). All PON proteins are able to hydrolyze lactones and PON1 is able to detoxify organophosphate pesticides and neurotoxins. Since neurotoxins are not normally present in the body the biological function of PON1 is thought to be protection of low-density lipoproteins from oxidation (Mackness et al., 1991). PON2 and PON3 share this function (Draganov et al., 2000; Ng et al., 2001). A higher incidence of ALS among Gulf war veterans and farmers suggested that chemical exposure may be a risk factor for ALS (Chió et al., 1991; Horner et al., 2003). Because PON proteins reduce oxidation and are able to detoxify neurotoxins these

Polymorphisms in *PON1* and *PON2* as well as a haploblock spanning *PON2* and *PON3* were found to be associated with sALS (Saeed et al., 2006; Slowik et al., 2006). Since then several other studies in different populations have reported association of SNPs in the *PON* genes with sALS (Cronin et al., 2007; Landers et al., 2008; Morahan et al., 2007; Valdmanis et al., 2008). However, a meta-analysis including 4037 cases and 4609 controls from five casecontrol studies and several genome-wide association studies showed no significant association between *PON* polymorphisms and ALS (Wills et al., 2009). More recently, two other studies failed to detect association between *PON* polymorphisms and ALS (Ricci et al. 2011; Zawislak et al., 2010). In a recent sequencing study, eight mutations in all three *PON* genes were identified in fALS and sALS patients (Ticozzi et al., 2010). Mutations in the *PON*

genes might play a role in ALS but additional sequencing is needed to confirm this.

Interestingly, PON1 activity can vary greatly depending on polymorphisms in its coding region (Costa et al., 2005). Thus, mutations in the *PON* genes could affect PON activity and thereby contribute to ALS pathogenesis. Toxicity in neurons caused by oxidative stress was higher in cells from PON2 knockout mice than in wild-type mice, suggesting that PON2 has a protective effect against neurotoxicity caused by oxidative stress (Giordano et al., 2011).

Peripherin is an intermediate filament similar to neurofilaments and is also associated with axonal spheroids in the proximal axon of spinal cord motor neurons of ALS patients (Corbo and Hays, 1992). It is also present in Lewy body-like inclusions and Bunina bodies that are seen in a portion of ALS patients (He and Hays, 2004; Mizuno et al., 2011). Peripherin is predominantly expressed in the peripheral nervous system and in spinal motor neurons in the central nervous system. After neuronal injury, peripherin expression is upregulated in spinal motor neurons and this upregulation has been linked to axonal regeneration (Troy et al., 1990). However, transgenic mice with wild-type overexpression of peripherin develop a late-onset and selective motor neuron disease characterized by intermediate filament inclusions (Beaulieu et al., 1999). For these reasons, the possibility of *PRPH* mutations in

*NEFH* gene in fALS and sALS samples (Vechio et al., 1996).

proteins have been investigated for association with ALS.

**3.6 Paraoxonase genes (PON)** 

**3.7 Peripherin (PRPH)** 

ALS patients was investigated. Two missense mutations and a frameshift deletion in the PRPH gene have been identified in sALS patients (Corrado et al., 2011; Gros-Louis et al., 2004; Leung et al., 2004). Additional screening of the *PRPH* gene for mutations in larger cohorts of ALS patients and controls is needed to determine the frequency and pathogenecity of *PRPH* mutations.

Expression of abnormal peripherin splice variants has also been suggested to play a role in ALS pathogenesis. A toxic splice variant of peripherin (Per61) was found in motor neurons of mutant SOD1 transgenic mice but not wild-type mice (Robertson et al., 2003). Expression of Per61 has more recently also been observed in mutant TDP-43 transgenic mice but not in wild-type TDP-43 transgenic mice (Swarup et al., 2011). In addition, Per61 specific antibodies stain aggregates in human ALS but not in control spinal cord (Swarup et al., 2011). The presence of abnormal peripherin splice variants (Per28) has also been shown in humans (Xiao et al., 2008). Per28 overexpression results in peripherin aggregation and an upregulation of peripherin expression at the mRNA and protein levels in ALS patients as compared to controls (Xiao et al., 2008). A different study showed expression of Per28 in lumbar spinal cord lysates of ALS patients but not control cases (McLean et al., 2010). Although the functional significance of these abnormal splice forms is unknown they seem to play a role in the development ALS.

### **3.8 Survival motor neuron (SMN) 1 and 2**

Two highly homologous copies of the survival motor neuron gene exist in humans, telomeric *SMN1* and centromeric *SMN2*. *SMN2*, which lacks exon 7 due to a nucleotide difference in a splice enhancer site, produces a less stable SMN protein and has only 20% of the biological function of SMN1 (Lorson et al., 1998). It has been shown that TDP-43 overexpression regulates the inclusion of exon 7 during pre-mRNA splicing of *SMN2* (Bose et al., 2008).

Deletions or mutations in *SMN1* cause the autosomal recessive disorder spinal muscular atrophy (SMA), whereas variation in *SMN2* copy number affects SMA disease severity (Lefebvre et al., 1997). SMA patients with a higher copy number of *SMN2* generally have a milder form of the disease (Gavrilov et al., 1998). SMN1 is widely expressed and functions in the assembly of the spliceosome as part of the SMN complex. SMN1 also interacts with several proteins involved in mRNA editing, transport, splicing, transcriptional regulation, and post-transcriptional processing and modification of rRNA (Eggert et al., 2006). The impaired assembly of the spliceosome could lead to neuronal degeneration.

Thus far, five different studies have failed to detect homozygous *SMN1* deletions in ALS patients (Gamez et al., 2002; Jackson et al., 1996; Moulard et al., 1998; Orrell et al., 1997; Parboosingh et al., 1999). However, an increased frequency of abnormal copy number (one or three copies) of *SMN1* was found in ALS patients compared to controls (Corcia et al., 2002). However, these results were inconsistent with other reports (Corcia et al., 2006; Veldink et al., 2001; Veldink et al., 2005). Recently, a large study was published including new samples of 847 sALS patients and 984 controls, showing that *SMN1* duplications were associated with ALS susceptibility (odds ratio [OR] = 2.07, 95% confidence interval [CI] = 1.34 - 3.20. (Blauw et al, 2011)). A meta-analysis of all previously published data, taking possible heterogeneity between studies into account, confirmed this association with *SMN1* duplications. Other work has shown that homozygous deletions of *SMN2* are associated

Genetics of Amyotrophic Lateral Sclerosis 493

Several genome-wide association studies (GWAS) have been performed in sALS patients. These studies have generated association results that have been replicated in the same study but rarely in independent studies. Although several of the associated genes discussed below are plausible to contribute to ALS considering their functional roles, the lack of consistent

A GWAS in 276 ALS patients and 271 healthy controls identified 34 possible associated SNPs but none of these reached genome-wide significance after Bonferroni correction (Schymick et al., 2007). A SNP near the gene FGGY carbohydrate kinase domain containing (*FGGY*) was reported to be associated in a GWAS in 1152 ALS patients with an odds ratio of 1.35 (Dunckley et al., 2007). However, two replication studies in a total of 2478 sALS patients and 2744 controls did not detect this association (Fernández-Santiago et al., 2011; Van Es et al., 2009b). No mutations in *FGGY* were found by sequencing in 190 ALS patients (Daoud et

A GWAS in 461 ALS patients and 450 controls found a variant in the inositol 1, 4, 5 triphosphate receptor 2 gene (*ITPR2*) to be associated with ALS. This association was replicated in the same study in a cohort of 876 patients and 906 controls and in the combined analysis (Van Es et al., 2007). ITPR2 has a role in glutamate-mediated neurotransmission, regulation of calcium concentration and apoptosis. However, the *ITPR2* association has not been found in a replication study and in subsequent GWAS (Chiò et al., 2009; Cronin et al., 2008; Fernández-Santiago et al., 2011; Laaksovirta et al., 2010; Shatunov et al., 2010; Van Es

Variation in the dipeptidyl-peptidase 6 (*DPP6*) gene was found to be significantly associated with sALS in a GWAS performed in a combined GWA data set from the USA and the Netherlands (Van Es et al., 2008). This association was replicated in three additional independent populations from The Netherlands, Sweden, and Belgium (Van Es et al., 2008). The same variant was the top hit in a joint analysis of GWA data sets in an Irish population and the same Dutch and American populations, although it did not reach genome-wide significance (Cronin et al., 2008). Upon addition of a Polish data set the association could not be replicated which could point to a population-specific effect (Cronin et al., 2009). In an Italian cohort of 266 ALS patients association of the same SNP was replicated (Del Bo et al., 2008b). However, subsequent replication studies and GWAS could not find evidence for a role of *DPP6* in ALS (Chiò et al., 2009; Daoud et al., 2010; Fogh et al., 2011; Laaksovirta et al., 2010; Li et al., 2009; Shatunov et al., 2010; Van Es et al., 2009c). Interestingly, in a genome scan for copy number variations, including 4434 ALS patients and over 14000 controls, a suggestive association was found for the *DPP6* locus (Blauw et al., 2010). Not much data is available on the function of DPP6, but it is expressed in brain and able to regulate the activity of neuropeptides and to bind A-type neuronal potassium channels (Nadal et al.,

Another two-stage GWAS in sALS patients was unable to find any associated SNPs that reached genome-wide significance, although suggestive association was found on

Survival analysis in a GWAS using samples from the USA and Europe revealed that a CC genotype of a SNP in the kinesin-associated protein 3 (*KIFAP3*) gene conferred a 14-month survival advantage on ALS patients (Landers et al., 2009). Expression data using RNA from brain tissue and lymphoblasts of patients showed that the favorable genotype significantly

**3.10 Genome wide association studies in sporadic ALS** 

al., 2010).

et al., 2009c).

2003).

**chromosome 7p13.3** (Chiò et al., 2009).

replication results makes it difficult to firmly establish their role in sALS.

with sporadic adult-onset lower motor neuron disease (Echaniz-Laguna et al., 2002; Moulard et al., 1998). Homozygous deletions of *SMN2* were also found to be overrepresented in 110 ALS patients (16%) compared to 100 controls (4%) (Veldink et al., 2001). *SMN2* deletions were associated with shorter survival in this study. However, a study by the same group using more ALS and control samples and several other studies did not find a higher frequency of *SMN2* deletions in ALS patients versus controls (Corcia et al., 2006; Gamez et al., 2002; Moulard et al., 1998; Parboosingh et al., 1999; Veldink et al., 2005). The recent meta-analysis showed that there is no increased frequency of homozygous *SMN2* deletions in ALS patients, and that neither *SMN1* nor *SMN2* appear to influence survival or age at onset of disease (Blauw et al. 2011).

Homozygous deletions in *SMN1* or *SMN2* do not play a role in ALS but an abnormal copy number in *SMN1* could increase risk for ALS and it is important to study the consequences on protein level in brain and spinal cord of having three copies of *SMN1* in order to determine the potential damaging effect.

#### **3.9 Vascular endothelial growth factor (VEGF)**

VEGF, a protein that stimulates angiogenesis in response to hypoxia, was identified as a candidate gene for ALS based on the finding that a deletion in the hypoxia response element (HRE) in the promoter of this gene in mice, resulting in decreased VEGF expression, led to progressive motor neuron degeneration (Oosthuyse et al., 2001). In addition, *VEGF* gene delivery in muscle and VEGF overexpression prolongs survival in mutant SOD1 transgenic mice. Furthermore, intracerebroventricular VEGF administration prolongs survival in mutant SOD1 transgenic rats (Azzouz et al., 2004; Storkebaum et al., 2005; Wang et al., 2007). Finally, decreased expression of VEGF and its receptor VEGFR2 is observed in spinal cords of ALS patients (Brockington et al., 2006).

Sequencing of the *VEGF* gene and its promotor in ALS patients failed to identify ALS specific mutations (Brockington et al., 2005; Gros-Luois et al., 2003; Lambrechts et al., 2003). However, a large study in 750 ALS patients and over 1200 controls from Sweden, Belgium, and England found association between two haplotypes determined by three SNPs and an increased risk for ALS (Lambrechts et al., 2003). These haplotypes lowered the circulating levels of VEGF and *VEGF* transcription (Lambrechts et al., 2003). This association was replicated in a study with small sample size (Terry et al., 2004). In contrast, subsequent studies could not confirm the association between *VEGF* and ALS in Dutch, British, American, Italian, Polish and Chinese populations (Brockington et al., 2005; Chen et al., 2006; Del Bo et al., 2008a; Golenia et al., 2010; Van Vught et al., 2005; Zhang et al., 2006). Furthermore, a meta-analysis on several of these studies found no association between *VEGF* polymorphisms and ALS (Lambrechts et al., 2009). A study in German ALS patients identified an association of a *VEGF* SNP with sALS in women (Fernández-Santiago et al., 2006). A different SNP was associated with ALS in male patients in a large meta-analysis (Lambrechts et al., 2009). This suggests that the role of *VEGF* in ALS may be gender dependent. An association of *VEGF* SNPs with age of onset in ALS was also reported although no such association was observed in the meta-analysis (Chen et al., 2007; Lambrechts et al., 2009).

In summary, studies in rodent models suggest a role for VEGF in ALS, possibly as a therapeutic target. However, genetic studies do not yet provide conclusive evidence for a genetic role for VEGF in ALS, although gender dependent effects may exist.

with sporadic adult-onset lower motor neuron disease (Echaniz-Laguna et al., 2002; Moulard et al., 1998). Homozygous deletions of *SMN2* were also found to be overrepresented in 110 ALS patients (16%) compared to 100 controls (4%) (Veldink et al., 2001). *SMN2* deletions were associated with shorter survival in this study. However, a study by the same group using more ALS and control samples and several other studies did not find a higher frequency of *SMN2* deletions in ALS patients versus controls (Corcia et al., 2006; Gamez et al., 2002; Moulard et al., 1998; Parboosingh et al., 1999; Veldink et al., 2005). The recent meta-analysis showed that there is no increased frequency of homozygous *SMN2* deletions in ALS patients, and that neither *SMN1* nor *SMN2* appear to influence survival or

Homozygous deletions in *SMN1* or *SMN2* do not play a role in ALS but an abnormal copy number in *SMN1* could increase risk for ALS and it is important to study the consequences on protein level in brain and spinal cord of having three copies of *SMN1* in order to

VEGF, a protein that stimulates angiogenesis in response to hypoxia, was identified as a candidate gene for ALS based on the finding that a deletion in the hypoxia response element (HRE) in the promoter of this gene in mice, resulting in decreased VEGF expression, led to progressive motor neuron degeneration (Oosthuyse et al., 2001). In addition, *VEGF* gene delivery in muscle and VEGF overexpression prolongs survival in mutant SOD1 transgenic mice. Furthermore, intracerebroventricular VEGF administration prolongs survival in mutant SOD1 transgenic rats (Azzouz et al., 2004; Storkebaum et al., 2005; Wang et al., 2007). Finally, decreased expression of VEGF and its receptor VEGFR2 is observed in spinal cords

Sequencing of the *VEGF* gene and its promotor in ALS patients failed to identify ALS specific mutations (Brockington et al., 2005; Gros-Luois et al., 2003; Lambrechts et al., 2003). However, a large study in 750 ALS patients and over 1200 controls from Sweden, Belgium, and England found association between two haplotypes determined by three SNPs and an increased risk for ALS (Lambrechts et al., 2003). These haplotypes lowered the circulating levels of VEGF and *VEGF* transcription (Lambrechts et al., 2003). This association was replicated in a study with small sample size (Terry et al., 2004). In contrast, subsequent studies could not confirm the association between *VEGF* and ALS in Dutch, British, American, Italian, Polish and Chinese populations (Brockington et al., 2005; Chen et al., 2006; Del Bo et al., 2008a; Golenia et al., 2010; Van Vught et al., 2005; Zhang et al., 2006). Furthermore, a meta-analysis on several of these studies found no association between *VEGF* polymorphisms and ALS (Lambrechts et al., 2009). A study in German ALS patients identified an association of a *VEGF* SNP with sALS in women (Fernández-Santiago et al., 2006). A different SNP was associated with ALS in male patients in a large meta-analysis (Lambrechts et al., 2009). This suggests that the role of *VEGF* in ALS may be gender dependent. An association of *VEGF* SNPs with age of onset in ALS was also reported although no such association was observed in the meta-analysis (Chen et al., 2007;

In summary, studies in rodent models suggest a role for VEGF in ALS, possibly as a therapeutic target. However, genetic studies do not yet provide conclusive evidence for a

genetic role for VEGF in ALS, although gender dependent effects may exist.

age at onset of disease (Blauw et al. 2011).

determine the potential damaging effect.

of ALS patients (Brockington et al., 2006).

Lambrechts et al., 2009).

**3.9 Vascular endothelial growth factor (VEGF)** 

### **3.10 Genome wide association studies in sporadic ALS**

Several genome-wide association studies (GWAS) have been performed in sALS patients. These studies have generated association results that have been replicated in the same study but rarely in independent studies. Although several of the associated genes discussed below are plausible to contribute to ALS considering their functional roles, the lack of consistent replication results makes it difficult to firmly establish their role in sALS.

A GWAS in 276 ALS patients and 271 healthy controls identified 34 possible associated SNPs but none of these reached genome-wide significance after Bonferroni correction (Schymick et al., 2007). A SNP near the gene FGGY carbohydrate kinase domain containing (*FGGY*) was reported to be associated in a GWAS in 1152 ALS patients with an odds ratio of 1.35 (Dunckley et al., 2007). However, two replication studies in a total of 2478 sALS patients and 2744 controls did not detect this association (Fernández-Santiago et al., 2011; Van Es et al., 2009b). No mutations in *FGGY* were found by sequencing in 190 ALS patients (Daoud et al., 2010).

A GWAS in 461 ALS patients and 450 controls found a variant in the inositol 1, 4, 5 triphosphate receptor 2 gene (*ITPR2*) to be associated with ALS. This association was replicated in the same study in a cohort of 876 patients and 906 controls and in the combined analysis (Van Es et al., 2007). ITPR2 has a role in glutamate-mediated neurotransmission, regulation of calcium concentration and apoptosis. However, the *ITPR2* association has not been found in a replication study and in subsequent GWAS (Chiò et al., 2009; Cronin et al., 2008; Fernández-Santiago et al., 2011; Laaksovirta et al., 2010; Shatunov et al., 2010; Van Es et al., 2009c).

Variation in the dipeptidyl-peptidase 6 (*DPP6*) gene was found to be significantly associated with sALS in a GWAS performed in a combined GWA data set from the USA and the Netherlands (Van Es et al., 2008). This association was replicated in three additional independent populations from The Netherlands, Sweden, and Belgium (Van Es et al., 2008). The same variant was the top hit in a joint analysis of GWA data sets in an Irish population and the same Dutch and American populations, although it did not reach genome-wide significance (Cronin et al., 2008). Upon addition of a Polish data set the association could not be replicated which could point to a population-specific effect (Cronin et al., 2009). In an Italian cohort of 266 ALS patients association of the same SNP was replicated (Del Bo et al., 2008b). However, subsequent replication studies and GWAS could not find evidence for a role of *DPP6* in ALS (Chiò et al., 2009; Daoud et al., 2010; Fogh et al., 2011; Laaksovirta et al., 2010; Li et al., 2009; Shatunov et al., 2010; Van Es et al., 2009c). Interestingly, in a genome scan for copy number variations, including 4434 ALS patients and over 14000 controls, a suggestive association was found for the *DPP6* locus (Blauw et al., 2010). Not much data is available on the function of DPP6, but it is expressed in brain and able to regulate the activity of neuropeptides and to bind A-type neuronal potassium channels (Nadal et al., 2003).

Another two-stage GWAS in sALS patients was unable to find any associated SNPs that reached genome-wide significance, although suggestive association was found on **chromosome 7p13.3** (Chiò et al., 2009).

Survival analysis in a GWAS using samples from the USA and Europe revealed that a CC genotype of a SNP in the kinesin-associated protein 3 (*KIFAP3*) gene conferred a 14-month survival advantage on ALS patients (Landers et al., 2009). Expression data using RNA from brain tissue and lymphoblasts of patients showed that the favorable genotype significantly

Genetics of Amyotrophic Lateral Sclerosis 495

<sup>1</sup>The

*Region fALS FTD ALS-*

15 N.K. N.K. N.K. N.K. 27 None

1 Scandinavia 5 9 - 2 None

America 1 - 9 14

6 France 9 10 12 29 + 4

(D9S169-D9S167) 1 Australia 2 5 2 11 None

(D9S235-D9S257) 1 Belgium 1 8 - 17 None

(D9S1808-D9S251) 1 USA 2 5 3 10 None

**et al. 2011** 9p21.2-p21.2 1 Wales 2 5 1 8 None

Fig. 1. Schematic overview of the associated regions found by linkage studies and GWAS. 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

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

Netherlands 7 2 3 3 None

France 14 3 4 4 None

*FTD* 

*Genes screened*  *Mutatio ns* 

p.Q342X in *IFT-74* 

miRNAs None

*Study Linkage region Families Country/* 

9p13.3-p22.1 (D9S1684- D9S1678)

9p13.2-p21.3 (D9S1870- D9S1791)

9p13.2-p21.2 (D9S2154- D9S1874)

9p13.2-p22.2

9p13.3-p22.2

9p11.2-p21.2 (AFM218xg11- D9S301)

9p21.2-q21

9p22.3-q21

9p21.2-p23

(D9S157-D9S1874) <sup>2</sup>North-

(D9S157-D9S1805) <sup>2</sup>Canada/

**Yan et al. 2006** 

**Morita et al. 2006** 

**Vance et al. 2006** 

**Momeni et al. 2006** 

**Valdmanis et al. 2007** 

**Le Ber et al. 2009** 

**Luty et al. 2008** 

**Boxer et al. 2010** 

**Pearson** 

**Gijselinck et al. 2010** 

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

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 *UNC13A* as being causative to sALS.

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