**2. Family-based linkage to ALSFTD2 locus on chromosome 9p**

Since the original reports of a Dutch and a Scandinavian ALS-FTLD family linked with chromosome 9p21 (Morita et al., 2006; Vance et al., 2006), a growing number of families with inherited ALS and FTLD are reported with significant linkage to the ALSFTD2 locus on chromosome 9p21 (Boxer et al., 2010; Gijselinck et al., 2010; Le Ber et al., 2009; Luty et al., 2008; Valdmanis et al., 2007) (table 1). In all these families patients show similar clinical and pathological characteristics. Clinically, individuals may present with symptoms of both ALS and FTLD, or with ALS or FTLD alone. Pathologically, autopsied patients have TDP-43 positive type 2 (Sampathu et al., 2006) brain inclusions (Boxer et al., 2010; Gijselinck et al., 2010; Le Ber et al., 2009; Luty et al., 2008; Morita et al., 2006; Vance et al., 2006). (table 1)

genetic heterogeneity is necessary to identify new therapeutic targets. Mutations causing ALS were observed in genes encoding Cu/Zn superoxide dismutase 1 (*SOD1*) (Rosen et al., 1993), TDP-43 (*TARDBP*) (Gitcho et al., 2008; Kabashi et al., 2008; Sreedharan et al., 2008; Van Deerlin et al., 2008; Yokoseki et al., 2008), fused in sarcoma (*FUS*) (Kwiatkowski, Jr. et al., 2009; Vance et al., 2009) and angiogenin (*ANG*) (Greenway et al., 2006), among other genes, while in familial FTLD patients mutations in the genes encoding granulin (*GRN*) (Baker et al., 2006; Cruts et al., 2006), the microtubule-associated protein tau (*MAPT*) (Hutton et al., 1998), the valosin-containing protein (*VCP*) (Watts et al., 2004) and the charged multivesicular body protein 2B (*CHMP2B*) (Skibinski et al., 2005) were found. Recent family-based linkage and population-based association studies identified genetic factors overlapping between ALS and FTLD. For example, mutations in the ALS genes *TARDBP* and *FUS* are occasionally found in FTLD patients (Kovacs et al., 2009; Van Langenhove et al., 2010) and mutations in the FTLD gene *VCP* were also detected in ALS (Johnson et al., 2010). However, most convincing evidence for the genetic overlap comes from the observation that both ALS and FTLD can occur within the same family or within a single patient of a family. More than 15 autosomal dominant families with ALS and FTLD worldwide are causally linked with a major disease locus at chromosome 9p13-p21 (ALSFTD2 locus) (Boxer et al., 2010; Gijselinck et al., 2010; Le Ber et al., 2009; Luty et al., 2008; Momeni et al., 2006; Morita et al., 2006; Pearson et al., 2011; Valdmanis et al., 2007; Vance et al., 2006). The minimally linked region in all these families is about 3.6 Mb in size containing five known protein-coding genes. Moreover, several recent genome-wide association studies (GWAS) in ALS populations from different European origins showed the presence of a major genetic risk factor for ALS at the same chromosome 9p region (Laaksovirta et al., 2010; Shatunov et al., 2010; van Es et al., 2009). The Finnish study narrowed the associated region to a 232 kb linkage disequilibrium (LD) block containing three known genes (*MOBKL2B*, *IFNK*, *C9orf72*) and suggested the presence of a major risk gene with high penetrance (Laaksovirta et al., 2010). Likewise, a GWAS in FTLD has implicated the same region (Van Deerlin et al., 2010). This finding was further confirmed in other FTLD and ALS-FTLD cohorts (Rollinson et al., 2011). Together, these data demonstrate that ALS and FTLD share a major common genetic factor on chromosome 9p, most likely showing high mutation frequencies. Despite all attempts of several research groups, the genetic defect(s) underlying both genetic linkage and association to this region have not

In this book chapter we will report and discuss the latest findings in the studies aiming at

Since the original reports of a Dutch and a Scandinavian ALS-FTLD family linked with chromosome 9p21 (Morita et al., 2006; Vance et al., 2006), a growing number of families with inherited ALS and FTLD are reported with significant linkage to the ALSFTD2 locus on chromosome 9p21 (Boxer et al., 2010; Gijselinck et al., 2010; Le Ber et al., 2009; Luty et al., 2008; Valdmanis et al., 2007) (table 1). In all these families patients show similar clinical and pathological characteristics. Clinically, individuals may present with symptoms of both ALS and FTLD, or with ALS or FTLD alone. Pathologically, autopsied patients have TDP-43 positive type 2 (Sampathu et al., 2006) brain inclusions (Boxer et al., 2010; Gijselinck et al., 2010; Le Ber et al., 2009; Luty et al., 2008; Morita et al., 2006; Vance et al., 2006). (table 1)

**2. Family-based linkage to ALSFTD2 locus on chromosome 9p** 

been identified yet.

identifying the chromosome 9 gene defect.

The minimal candidate region was previously defined by D9S169 (Luty et al., 2008) and D9S1805 (Valdmanis et al., 2007) spanning 7 Mb and was recently reduced to 3.6 Mb between D9S169 (Luty et al., 2008) and D9S251 by Boxer and colleagues (2010) (figure 1). Therefore, several parts of this study were still investigated in the 7 Mb region.


Table 1. Genetic, clinical and pathological characteristics of ALS-FTLD families linked or associated with chromosome 9p21 (ND: not determined; 1summed LODscore in 6 small families, not linked separately)

A Major Genetic Factor at Chromosome 9p Implicated in

**2.1 Family DR14** 

Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD) 541

We studied a Belgian 4-generation family (family DR14) with autosomal dominant transmission of ALS and FTLD. We collected DNA from 29 family members of whom 3 patients in generation III and 11 at-risk individuals in generation III and IV each (figure 1). Two patients (III.2 and III.12) were diagnosed with FTLD (subtype FTD), while patient III.10 was diagnosed with ALS (figure 2). The mean age at onset was 58.1 4.2 years (N = 9, range 51-65 years) and mean disease duration was 6.4 4.9 years (range 1-17 years). The index patient was clinically diagnosed with familial FTLD (subtype FTD) and pathological TDP-43 positive inclusions were observed in the brain (FTLD-TDP type 2 (Sampathu et al., 2006)). Mutations in the known genes for ALS and dementia were excluded. (Gijselinck et al., 2010)

Fig. 2. The pedigree of DR14 consists of four generations. Left-filled and right-filled symbols represent patients with FTLD and ALS respectively. Patients with unspecified dementia are indicated with filled symbols. Open symbols represent unaffected individuals or at-risk individuals with unknown phenotype. Individuals with unclear phenotype are designated with a question mark (?). The arrow indicates index patient III.12. Numbers below the symbols denote age at onset and age at death (AAD) for patients and either age at last examination (AALE) or AAD for unaffected individuals or individuals with unknown

phenotype. An asterisk (\*) indicates individuals of whom DNA was available.

**2.2 Mutation analyses of known genes and conserved regions, and CNV analysis**  We performed a genome-wide scan using an in-house developed mapping set of 425 microsatellite markers in 30 multiplex panels with an average distance of 8 cM. Multipoint LOD scores were calculated revealing two loci on chromosome 9 and 14: one at chromosome 9 with a maximal LOD score of 2.71 between D9S1121 and D9S270 and one at chromosome 14 with the highest LOD score of 2.61 between D14S302 and D14S611. Finemapping of the chromosome 9 locus resulted in a significant maximal multipoint LOD score of 3.38 between D9S1833 and D9S1121 at 9p21 and segregation analysis defined a candidate region of 64.6 cM (74.7 Mb) between markers D9S235 and D9S257 on chromosome 9p23-9q21, based on two obligate recombinants (figure 3), harboring 271 protein coding genes (Gijselinck et al., 2010). This region overlaps with the ALSFTD2 locus at chromosome 9p21 but did not reduce the minimally linked region. Therefore, we analyzed the 7 Mb overlap region, including the minimal locus of 3.6 Mb, for mutations. We sequenced all 27 protein-coding genes, either the complete coding sequence of cDNA (N=17) including *MOBKL2B*, *C9orf72*, *ACO1*, *DDX58*, *TOPORS*, *NDUFB6*, *DNAJA1*, *SMU1*, *B4GALT1*, *BAG1*, *CHMP5*, *AQP3*, *NOL6*, *UBE2R2*, *UBAP2*, *WDR40A* and *UBAP1*, or the exons and exon-intron boundaries on gDNA using classical sequencing (N=10). cDNA was prepared from lymphoblasts of two patients and two healthy control individuals of the family not carrying the disease haplotype, treated with or without cycloheximide allowing also the detection of degraded aberrant transcripts.

Fig. 1. Schematic representation of the chromosome 9p21 ALS-FTLD locus. Upper panel: grey bars indicate the minimal candidate regions in all reported significantly linked ALS-FTLD families, defining a minimal interval of 3.7 Mb between D9S169 and D9S251 containing five protein coding genes, illustrated with grey lines. Lower panel: associated SNPs in ALS and FTLD GWAS are shown in red and LD blocks or finemapped regions of these GWAS are indicated with green lines. Three genes are located in the associated region.

### **2.1 Family DR14**

540 Amyotrophic Lateral Sclerosis

Fig. 1. Schematic representation of the chromosome 9p21 ALS-FTLD locus. Upper panel: grey bars indicate the minimal candidate regions in all reported significantly linked ALS-FTLD families, defining a minimal interval of 3.7 Mb between D9S169 and D9S251 containing five protein coding genes, illustrated with grey lines. Lower panel: associated SNPs in ALS and FTLD GWAS are shown in red and LD blocks or finemapped regions of these GWAS are indicated with green lines. Three genes are located in the associated region. We studied a Belgian 4-generation family (family DR14) with autosomal dominant transmission of ALS and FTLD. We collected DNA from 29 family members of whom 3 patients in generation III and 11 at-risk individuals in generation III and IV each (figure 1). Two patients (III.2 and III.12) were diagnosed with FTLD (subtype FTD), while patient III.10 was diagnosed with ALS (figure 2). The mean age at onset was 58.1 4.2 years (N = 9, range 51-65 years) and mean disease duration was 6.4 4.9 years (range 1-17 years). The index patient was clinically diagnosed with familial FTLD (subtype FTD) and pathological TDP-43 positive inclusions were observed in the brain (FTLD-TDP type 2 (Sampathu et al., 2006)). Mutations in the known genes for ALS and dementia were excluded. (Gijselinck et al., 2010)

Fig. 2. The pedigree of DR14 consists of four generations. Left-filled and right-filled symbols represent patients with FTLD and ALS respectively. Patients with unspecified dementia are indicated with filled symbols. Open symbols represent unaffected individuals or at-risk individuals with unknown phenotype. Individuals with unclear phenotype are designated with a question mark (?). The arrow indicates index patient III.12. Numbers below the symbols denote age at onset and age at death (AAD) for patients and either age at last examination (AALE) or AAD for unaffected individuals or individuals with unknown phenotype. An asterisk (\*) indicates individuals of whom DNA was available.

### **2.2 Mutation analyses of known genes and conserved regions, and CNV analysis**

We performed a genome-wide scan using an in-house developed mapping set of 425 microsatellite markers in 30 multiplex panels with an average distance of 8 cM. Multipoint LOD scores were calculated revealing two loci on chromosome 9 and 14: one at chromosome 9 with a maximal LOD score of 2.71 between D9S1121 and D9S270 and one at chromosome 14 with the highest LOD score of 2.61 between D14S302 and D14S611. Finemapping of the chromosome 9 locus resulted in a significant maximal multipoint LOD score of 3.38 between D9S1833 and D9S1121 at 9p21 and segregation analysis defined a candidate region of 64.6 cM (74.7 Mb) between markers D9S235 and D9S257 on chromosome 9p23-9q21, based on two obligate recombinants (figure 3), harboring 271 protein coding genes (Gijselinck et al., 2010). This region overlaps with the ALSFTD2 locus at chromosome 9p21 but did not reduce the minimally linked region. Therefore, we analyzed the 7 Mb overlap region, including the minimal locus of 3.6 Mb, for mutations. We sequenced all 27 protein-coding genes, either the complete coding sequence of cDNA (N=17) including *MOBKL2B*, *C9orf72*, *ACO1*, *DDX58*, *TOPORS*, *NDUFB6*, *DNAJA1*, *SMU1*, *B4GALT1*, *BAG1*, *CHMP5*, *AQP3*, *NOL6*, *UBE2R2*, *UBAP2*, *WDR40A* and *UBAP1*, or the exons and exon-intron boundaries on gDNA using classical sequencing (N=10). cDNA was prepared from lymphoblasts of two patients and two healthy control individuals of the family not carrying the disease haplotype, treated with or without cycloheximide allowing also the detection of degraded aberrant transcripts.

A Major Genetic Factor at Chromosome 9p Implicated in

in mammals e.g. between human and mouse (Ravasi et al., 2010).

2010). Cytogenetics excluded large chromosomal rearrangements.

in the linked region.

**2.3 Whole genome sequencing** 

addition to single nucleotide polymorphisms (SNPs).

Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD) 543

approach, we excluded mutations in highly conserved regions. However, we did not exclude variants in regions with no or low conservation in mammalian species because it is well known that a substantial number of primate/human-specific exons exist (e.g. Sela et al., 2007) and that the location of regulatory elements is not always highly conserved, even not

In addition, we performed chromosome-specific oligo-based array-comparative genomic hybridization (array-CGH, Nimblegen) at chromosome 9 with a resolution of about 1kb, on the index patient and an independent control individual not carrying the disease haplotype to detect copy number variations (CNVs). The CGH data were analyzed by Signalmap software (Nimblegen) and the scoring program CGHcall, revealing one large CNV (chr9:29082732-29087816) covered by 20 CGH probes. This deletion was confirmed in the index patient by six qPCR fragments demonstrating a deleted region of at least 5273 bp (chr9:29082677-29087949) (data not shown). It did not segregate with disease in DR14 and represented a polymorphism since it was also present in individuals not carrying the disease haplotype and since a frequent CNV had previously been reported at this position (chr9:29082445-29088195) (Cooper et al., 2008). Consequently, these experiments failed to identify a copy number mutation (deletion or insertion) of more than 1 kb (Gijselinck et al.,

Since all these mutation analyses did not reveal the causal mutation, we hypothesized that the mutation is most likely unusual with respect to location (extragenic or intronic) and/or type (small indel, inversion or other complex rearrangement). Therefore, we performed whole genome sequencing in family DR14 and subsequently analyzed sequences or variants

The complete genome sequence of four chromosome 9p disease haplotype carriers of family DR14, including two patients and two asymptomatic individuals was determined using next generation sequencing technology. These family members were selected such that they have a different unaffected haplotype. The sequencing was done with the company Complete Genomics (Mountain View CA, USA, www.completegenomics.com) who provides 35 bp paired-end sequence reads at a high sequence coverage obtained with high-accuracy combinatorial probe anchor ligation (cPAL) sequencing technology (Drmanac et al., 2010; Roach et al., 2010). Also, the paired-end sequencing data enable the identification of copy number variations (CNVs) and other structural variants (SV) including inversions, in

In the 4 genomes, we obtained an average coverage of 62-fold genome sequence and captured both alleles at 95.4% of the genomes. All sequence variants, including SNPs and small indels, were mapped to the human reference genome sequence (NCBI Build 36/hg18). We initially focused on the 3.6 Mb candidate region on chromosome 9p21. We filtered and prioritized variants according to several criteria. First, variants must be present heterozygously in all 4 patients since the disease is segregating in an autosomal dominant manner. As a heterozygous variant might be rarely missed using NGS technology, depending on local sequence coverage and quality, variants detected in three of four patients were also considered. Second, variants were selected that were not catalogued in the dbSNP database (http://www.ncbi.nlm.nih.gov/projects/SNP) and were not found as common polymorphisms (allele frequency ≥ 1%) in the 1000 Genomes

Mutation analysis on cDNA allows not only detecting simple point mutations and small insertions/deletions but also exon deletions/duplications and alternative transcripts. Similar to other chr9-linked ALS-FTLD families, this mutation analysis did not reveal patient-specific novel variants segregating with disease.

Fig. 3. Segregation of the 9p23-q21 haplotype in family DR14. Haplotypes are based on a selection of 20 informative STR markers at chromosome 9. The black haplotype represents the disease haplotype. Haplotypes for deceased individuals were inferred based on genotype data obtained in their offspring (between brackets). The disease haplotype was arbitrarily set for I.1, and numbers in diamonds indicate the number of genotyped at-risk individuals. An asterisk (\*) indicates individuals of whom DNA was available.

Since all coding exons of known genes were excluded for mutations, we selected other evolutionary conserved regions and investigated these sequences for the presence of noncoding variants in evolutionary constrained regulatory elements, e.g. promoters and distant regulatory elements or conserved epigenetic sequence motifs, or coding variants in unknown novel genes (protein coding or non-coding RNA genes). Using the UCSC-PhastCons-mammalian-28way track predicting and scoring the presence of conserved elements in the genome by comparing the sequence between 28 mammalian species, we defined 149 kb of conserved elements throughout the ALSFTD2 locus of 7 Mb. These elements were grouped in 1108 clusters with a total sequence of 465 kb and ranked according to conservation strength. We performed sanger sequencing in two patients and two healthy control individuals of the family not carrying the disease haplotype. In total we sequenced 95 kb of highest conserved elements (total of 260 kb clusters) in the 7 Mb region, not revealing patient-specific novel variants segregating with disease. Of these, 61 kb of conserved regions are located in the minimal candidate region of 3.6 Mb. Using this approach, we excluded mutations in highly conserved regions. However, we did not exclude variants in regions with no or low conservation in mammalian species because it is well known that a substantial number of primate/human-specific exons exist (e.g. Sela et al., 2007) and that the location of regulatory elements is not always highly conserved, even not in mammals e.g. between human and mouse (Ravasi et al., 2010).

In addition, we performed chromosome-specific oligo-based array-comparative genomic hybridization (array-CGH, Nimblegen) at chromosome 9 with a resolution of about 1kb, on the index patient and an independent control individual not carrying the disease haplotype to detect copy number variations (CNVs). The CGH data were analyzed by Signalmap software (Nimblegen) and the scoring program CGHcall, revealing one large CNV (chr9:29082732-29087816) covered by 20 CGH probes. This deletion was confirmed in the index patient by six qPCR fragments demonstrating a deleted region of at least 5273 bp (chr9:29082677-29087949) (data not shown). It did not segregate with disease in DR14 and represented a polymorphism since it was also present in individuals not carrying the disease haplotype and since a frequent CNV had previously been reported at this position (chr9:29082445-29088195) (Cooper et al., 2008). Consequently, these experiments failed to identify a copy number mutation (deletion or insertion) of more than 1 kb (Gijselinck et al., 2010). Cytogenetics excluded large chromosomal rearrangements.

Since all these mutation analyses did not reveal the causal mutation, we hypothesized that the mutation is most likely unusual with respect to location (extragenic or intronic) and/or type (small indel, inversion or other complex rearrangement). Therefore, we performed whole genome sequencing in family DR14 and subsequently analyzed sequences or variants in the linked region.

### **2.3 Whole genome sequencing**

542 Amyotrophic Lateral Sclerosis

Mutation analysis on cDNA allows not only detecting simple point mutations and small insertions/deletions but also exon deletions/duplications and alternative transcripts. Similar to other chr9-linked ALS-FTLD families, this mutation analysis did not reveal

Fig. 3. Segregation of the 9p23-q21 haplotype in family DR14. Haplotypes are based on a selection of 20 informative STR markers at chromosome 9. The black haplotype represents the disease haplotype. Haplotypes for deceased individuals were inferred based on genotype data obtained in their offspring (between brackets). The disease haplotype was arbitrarily set for I.1, and numbers in diamonds indicate the number of genotyped at-risk

Since all coding exons of known genes were excluded for mutations, we selected other evolutionary conserved regions and investigated these sequences for the presence of noncoding variants in evolutionary constrained regulatory elements, e.g. promoters and distant regulatory elements or conserved epigenetic sequence motifs, or coding variants in unknown novel genes (protein coding or non-coding RNA genes). Using the UCSC-PhastCons-mammalian-28way track predicting and scoring the presence of conserved elements in the genome by comparing the sequence between 28 mammalian species, we defined 149 kb of conserved elements throughout the ALSFTD2 locus of 7 Mb. These elements were grouped in 1108 clusters with a total sequence of 465 kb and ranked according to conservation strength. We performed sanger sequencing in two patients and two healthy control individuals of the family not carrying the disease haplotype. In total we sequenced 95 kb of highest conserved elements (total of 260 kb clusters) in the 7 Mb region, not revealing patient-specific novel variants segregating with disease. Of these, 61 kb of conserved regions are located in the minimal candidate region of 3.6 Mb. Using this

individuals. An asterisk (\*) indicates individuals of whom DNA was available.

patient-specific novel variants segregating with disease.

The complete genome sequence of four chromosome 9p disease haplotype carriers of family DR14, including two patients and two asymptomatic individuals was determined using next generation sequencing technology. These family members were selected such that they have a different unaffected haplotype. The sequencing was done with the company Complete Genomics (Mountain View CA, USA, www.completegenomics.com) who provides 35 bp paired-end sequence reads at a high sequence coverage obtained with high-accuracy combinatorial probe anchor ligation (cPAL) sequencing technology (Drmanac et al., 2010; Roach et al., 2010). Also, the paired-end sequencing data enable the identification of copy number variations (CNVs) and other structural variants (SV) including inversions, in addition to single nucleotide polymorphisms (SNPs).

In the 4 genomes, we obtained an average coverage of 62-fold genome sequence and captured both alleles at 95.4% of the genomes. All sequence variants, including SNPs and small indels, were mapped to the human reference genome sequence (NCBI Build 36/hg18). We initially focused on the 3.6 Mb candidate region on chromosome 9p21. We filtered and prioritized variants according to several criteria. First, variants must be present heterozygously in all 4 patients since the disease is segregating in an autosomal dominant manner. As a heterozygous variant might be rarely missed using NGS technology, depending on local sequence coverage and quality, variants detected in three of four patients were also considered. Second, variants were selected that were not catalogued in the dbSNP database (http://www.ncbi.nlm.nih.gov/projects/SNP) and were not found as common polymorphisms (allele frequency ≥ 1%) in the 1000 Genomes

A Major Genetic Factor at Chromosome 9p Implicated in

**3.1 Replication study chr9p21 GWAS** 

different populations.

Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD) 545

We investigated association of the most widely studied GWAS top SNP at chr9p21, rs2814707, in a Flanders-Belgian population of genealogically unrelated patients clinically diagnosed with ALS (N=124), ALS-FTLD (N=21) or FTLD (N=203) according to established consensus criteria (Brooks et al., 2000; Neary et al., 1998), compared to a group of 510 unrelated neurologically healthy control individuals from the same region in Belgium. We genotyped rs2814707 and showed that this SNP is in Hardy-Weinberg Equilibrium. Allelic and genotypic single SNP association was calculated using logistic regression analysis. The SNP showed significant allelic and genotypic association in the total population and highly significant association in the ALS and ALS-FTLD subpopulation reaching a maximal odds ratio of 3.27 in ALS patients homozygous for the minor allele (table 2). In the FTLD subpopulation no association was found, demonstrating that the effect in the total population can entirely be explained by the effect in patients with an ALS phenotype. When we include 21 ALS samples of Bulgarian origin, the relative risk became even higher, compared to Belgians only, indicating that the associated allele is the same between

**SNP ID Genotype Controls ALS, ALS-FTLD and FTLD ALS and ALS-FTLD** 

**freq (%) freq (%) p-value OR (95%CI) freq (%) p-value OR (95%CI)** 

rs2814707 C 77.9 71.3 **0.006** 1.39 (1.10-1.75) 67.0 **0.001** 1.69 (1.25-2.29)

CC 59.7 50.4 ref ref 43.1 ref ref

Table 2. Allelic and genotypic association of a GWAS top SNP in the total population and the ALS/ALS-FTLD subpopulation. P-values are corrected for age at onset or inclusion and

We combined the data from the different GWA studies and our study to determine the relative risk of carrying the risk allele on chromosome 9p21. A meta-analysis of the most widely studied SNP on chromosome 9p21 (rs2814707) underscores the presence of a genetic risk factor for ALS and/or FTLD at this locus. Carriers of the rs2814707 minor allele are at increased risk to develop ALS or FTLD (ORmeta 1.29 (95% CI 1.18-1.41), p-value 2.3\*10-8 (Figure 4)). When excluding the GWAS cohorts in which the association was first reported (van Es et al., 2009) to exclude bias because of winner's curse, the strength of the association remains similar (ORmeta 1.32 (95%CI 1.17-1.49; p-value 3.5\*10-6). Exclusion of three studies, including our own, which combine FTLD and ALS phenotypes would have resulted in an

 CT 36.5 41.8 0.08 1.31 (0.97-1.77) 47.9 **0.007** 1.76 (1.17-2.64) TT 3.8 7.8 **0.008** 2.41 (1.26-4.62) 9.0 **0.005** 3.27 (1.44-7.41)

**N=510 N=348 N=145** 

T 22.1 28.7 33.0

gender. (OR: odds ratio; CI: confidence interval)

ORmeta 1.24 (95%CI 1.13-1.36); p-value 3.3\*10-6).

**3.2 Meta analysis on chromosome 9p21** 

Project (http://www.1000genomes.org). Third, variants in nucleotide stretches were filtered out because they are known to be error-prone in NGS data. This resulted in a total of 189 variants, all located outside coding regions of known genes confirming genebased mutation analyses. These variants were genotyped in all 29 individuals of the DR14 family using Sanger sequencing and tested for segregation. 120 variants were located on the disease haplotype and were analyzed in a series of 300 neurologically healthy control individuals collected in Flanders, Belgium, i.e. the geographical region of which family DR14 originates, using multiplex Sequenom MassARRAY technology. 37 of these variants were completely absent in 300 control individuals and are all located in untranslated regions or introns of genes, or intergenic. We are currently prioritizing these variants based on evolutionary conservation, regulatory potential, location compatible with cis-acting function on functional candidate genes, etc. Also, we are determining the presence of these variants in a Belgian population of unrelated patients with ALS (N=124), ALS-FTLD (N=21) and FTLD (N=203), aiming to find a possible founder mutation. We already showed evidence for the presence of founder mutations in the Flanders-Belgian FTLD collection, by the *GRN* IVS1+5 G>C founder mutation identified in 19% of familial FTLD (Cruts et al., 2006). We have investigated the patient population for chromosome 9p STR markers and did not find evidence for haplotype sharing with family DR14; however, we cannot exclude the presence of a small, previously undetected founder haplotype.
