**4. Discussion and conclusion**

Family-based linkage and population-based association studies in Belgian patients with ALS and/or FTLD provided further evidence for the presence of a major genetic factor on chromosome 9p21 for these diseases.

In the Belgian family DR14 we analyzed the minimally linked region shared in all linked families. We excluded mutations in exons of all known protein-coding genes, in the highest conserved sequences and also copy number mutations of more than 1 kb were excluded. Further we used next generation sequencing technology to sequence the whole genome of four disease haplotype carriers. We are currently analyzing the first selection of variants. If we are left with only a very small number of putative disease-associated variants, we will analyze the complete sequence of the functional unit in which the remaining variants are located in the complete set of ALS, ALS-FTLD and FTLD patients. `Functional unit' in this

Fig. 4. Forest plot of a random effects meta-analysis of rs2814707. Meta-analysis was conducted in rmeta v2.16, and based on effect estimates and standard errors for the minor allele reported in each individual publication. Odds Ratios and 95% Confidence Intervals are given for each study separately along with a summary Odds Ratio, of the minor allele relative to the major allele. All 9p21 association studies on ALS, ALS-FTLD and FTLD published until July 2011 were included, in addition to our own unpublished data. For the study of Shatunov and colleagues we only included data on the independent UK cohort, to avoid overlap of datasets with previous studies. From Rollinson et al, only data on the

Family-based linkage and population-based association studies in Belgian patients with ALS and/or FTLD provided further evidence for the presence of a major genetic factor on

In the Belgian family DR14 we analyzed the minimally linked region shared in all linked families. We excluded mutations in exons of all known protein-coding genes, in the highest conserved sequences and also copy number mutations of more than 1 kb were excluded. Further we used next generation sequencing technology to sequence the whole genome of four disease haplotype carriers. We are currently analyzing the first selection of variants. If we are left with only a very small number of putative disease-associated variants, we will analyze the complete sequence of the functional unit in which the remaining variants are located in the complete set of ALS, ALS-FTLD and FTLD patients. `Functional unit' in this

Manchester ALS-FTLD cohort are included.

**4. Discussion and conclusion** 

chromosome 9p21 for these diseases.

context means the gene, regulatory element, conserved element or, in the absence of recognizable elements, 1 kb flanking each side of the putative mutation. This might identify additional mutations resulting in the same functional defect as the mutations detected in DR14 and further enhance the likelihood of the variant(s) to be disease-related. Finding such variants will provide strong genetic evidence of a disease causing effect of the variants. Alternatively, in case we do not find a mutation in this first selection of variants, we can use more relaxing filters. Taking into account that dbSNP may include rare clinical variants, rare or non-validated dbSNP SNPs will also be considered (N=91). Also the candidate region can be extended to the next recombinant or to the large DR14 candidate region. Further, regions that are not covered in more than one genome, will be completed using classical sanger sequencing. Finally, structural variants and copy number mutations will be investigated.

More than five years of research in the ALSFTD2 locus in different ALS-FTLD families worldwide did not identify pathogenic mutations yet (table 1), although mutations in two different genes on chromosome 9 outside the minimal candidate region, *IFT74* and *SIGMAR1*, were suggested (Luty et al., 2010; Momeni et al., 2006) but without further confirmation in other families. The fact that the culprit gene is still not found may in part be explained by the fact that families linked with chromosome 9p21 do not all have the same disease haplotype so that different mutations, probably with the same effect on the same gene, are most likely involved. Also, the causal mutations are most likely unusual with respect to position or type. For example, deep intronic mutations or mutations in a distant regulatory element might cause the disease but assessing their effect is rather complicated. Also, identification of small insertions/deletions or inversions is challenging.

In addition, we replicated association in a Belgian cohort of ALS, ALS-FTLD and FTLD patients of two major top SNPs on chromosome 9p21 previously associated in several ALS and FTLD GWA studies. More specifically, we found that the risk haplotype at chromosome 9p21 is most substantially increased in patients with ALS or ALS-FTLD compared to control individuals. The lack of association in the FTLD subpopulation is similar to what was observed in a previous replication study in which association was only found in ALS-FTLD patients (Rollinson et al., 2011). Also, the weakest association signal was found in the FTLD GWAS compared to ALS GWAS. This is the first time that a susceptibility locus for ALS is replicated in different GWA studies and replication studies, underlining the importance of the chromosome 9p21 locus harbouring a risk increasing factor for ALS (and ALS-FTLD) across multiple populations with a high relative risk of disease susceptibility. We are further characterizing this genetic association to reduce the associated region in the Belgian population. We are finemapping the chromosome 9p risk haplotype in great detail in our ALS, FTLD, ALS-FTLD patient cohorts by making a high density SNP map of the complete LD block and using extended association analyses of series of known and newly identified variants in the LD block. These variants were identified in previous publications, hapmap, 1000 Genomes Project and extended genomic sequencing efforts of the linkage disequilibrium block in a selection of ALS and ALS-FTLD patients carrying the associated allele of the GWAS SNPs in a homozygous or heterozygous state. This will finally result in the identification of the functional variant explaining the strong association in the chromosome 9p21 region.

The observation that the chromosome 9p21 region is harboring both disease-causing variants and susceptibility factors with high penetrance, might suggest that different genetic variants with variable degree of biological consequences might be involved. Alternatively,

A Major Genetic Factor at Chromosome 9p Implicated in

*Other Motor Neuron Disord*, 1, 5, 293-299

to chromosome 17q21. *Nature*, 442, 7105, 920-924

self-assembling DNA nanoarrays. *Science*, 327, 5961, 78-81

*Nat Genet*, 40, 10, 1199-1203

*Neurol*, 67, 5, 606-616

*Psychiatry* 

Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD) 549

Baker, M., Mackenzie, I.R., Pickering-Brown, S.M., Gass, J., Rademakers, R., Lindholm, C.,

Brooks, B.R., Miller, R.G., Swash, M. & Munsat, T.L. (2000). El Escorial revisited: revised

Cooper, G.M., Zerr, T., Kidd, J.M., Eichler, E.E. & Nickerson, D.A. (2008). Systematic

Cruts, M., Gijselinck, I., van der Zee, J., Engelborghs, S., Wils, H., Pirici, D., Rademakers, R.,

Drmanac, R., Sparks, A.B., Callow, M.J., Halpern, A.L., Burns, N.L., Kermani, B.G.,

Gijselinck, I., Engelborghs, S., Maes, G., Cuijt, I., Peeters, K., Mattheijssens, M., Joris, G.,

Gitcho, M.A., Baloh, R.H., Chakraverty, S., Mayo, K., Norton, J.B., Levitch, D., Hatanpaa,

mutation in familial motor neuron disease. *Ann Neurol*, 63, 4, 535-538

Cras, P., Martin, J.J., De Deyn, P.P., Kumar-Singh, S., Van Broeckhoven, C. & Cruts, M. (2010). Identification of 2 Loci at chromosomes 9 and 14 in a multiplex family with frontotemporal lobar degeneration and amyotrophic lateral sclerosis. *Arch* 

K.J., White, C.L., III, Bigio, E.H., Caselli, R., Baker, M., Al Lozi, M.T., Morris, J.C., Pestronk, A., Rademakers, R., Goate, A.M. & Cairns, N.J. (2008). TDP-43 A315T

Snowden, J., Adamson, J., Sadovnick, A.D., Rollinson, S., Cannon, A., Dwosh, E., Neary, D., Melquist, S., Richardson, A., Dickson, D., Berger, Z., Eriksen, J., Robinson, T., Zehr, C., Dickey, C.A., Crook, R., McGowan, E., Mann, D., Boeve, B., Feldman, H. & Hutton, M. (2006). Mutations in progranulin cause tau-negative frontotemporal dementia linked to chromosome 17. *Nature*, 442, 7105, 916-919 Boxer, A.L., Mackenzie, I.R., Boeve, B.F., Baker, M., Seeley, W.W., Crook, R., Feldman, H.,

Hsiung, G.Y., Rutherford, N., Laluz, V., Whitwell, J., Foti, D., McDade, E., Molano, J., Karydas, A., Wojtas, A., Goldman, J., Mirsky, J., Sengdy, P., Dearmond, S., Miller, B.L. & Rademakers, R. (2010). Clinical, neuroimaging and neuropathological features of a new chromosome 9p-linked FTD-ALS family. *J Neurol Neurosurg* 

criteria for the diagnosis of amyotrophic lateral sclerosis. *Amyotroph Lateral Scler* 

assessment of copy number variant detection via genome-wide SNP genotyping.

Vandenberghe, R., Dermaut, B., Martin, J.J., van Duijn, C., Peeters, K., Sciot, R., Santens, P., de Pooter, T., Mattheijssens, M., Van den Broeck, M., Cuijt, I., Vennekens, K., De Deyn, P.P., Kumar-Singh, S. & Van Broeckhoven, C. (2006). Null mutations in progranulin cause ubiquitin-positive frontotemporal dementia linked

Carnevali, P., Nazarenko, I., Nilsen, G.B., Yeung, G., Dahl, F., Fernandez, A., Staker, B., Pant, K.P., Baccash, J., Borcherding, A.P., Brownley, A., Cedeno, R., Chen, L., Chernikoff, D., Cheung, A., Chirita, R., Curson, B., Ebert, J.C., Hacker, C.R., Hartlage, R., Hauser, B., Huang, S., Jiang, Y., Karpinchyk, V., Koenig, M., Kong, C., Landers, T., Le, C., Liu, J., McBride, C.E., Morenzoni, M., Morey, R.E., Mutch, K., Perazich, H., Perry, K., Peters, B.A., Peterson, J., Pethiyagoda, C.L., Pothuraju, K., Richter, C., Rosenbaum, A.M., Roy, S., Shafto, J., Sharanhovich, U., Shannon, K.W., Sheppy, C.G., Sun, M., Thakuria, J.V., Tran, A., Vu, D., Zaranek, A.W., Wu, X., Drmanac, S., Oliphant, A.R., Banyai, W.C., Martin, B., Ballinger, D.G., Church, G.M. & Reid, C.A. (2010). Human genome sequencing using unchained base reads on

one genetic defect might act as high penetrant susceptibility factor in sporadic patients and as disease-causing factor with reduced penetrance in ALS-FTLD families, carrying also other disease modifying factors. In this respect it is interesting to note that in our studied belgian family DR14 all patients carry in addition to the disease haplotype at chromosome 9p21 also a haplotype in a novel locus at chromosome 14q32, possibly harboring a disease modifying gene (Gijselinck et al., 2010) and of which the sequences are present in the whole genome sequencing data of the family. Combining the family-based and the population-based approach to ultimately find the gene with one or more genetic defects would be of great value. For example, prioritizing the associated LD block in the whole genome sequence analysis of the family could be useful. Further, since in the associated LD block only three genes are located (*IFNK*, *C9orf72*, *MOBKL2B*) (figure 1), we could focus on these genes with respect to expression and dosage studies (eg. single exon deletions or duplications) in the family. Also, the region in and around the associated LD block can be saturated with STR markers for sharing studies with the DR14 family to detect a small founder haplotype. Combining all these comprehensive data will bring us closer to the identification of the chromosome 9 gene. As long as the genetic defect underlying linkage and association is not known, the full epidemiological impact of the chromosome 9p gene in familial and nonfamilial forms of ALS, ALS-FTLD and FTLD cannot be determined. However, the combined evidence emerging from all molecular genetic studies in chromosome 9p21-linked families and in chromosome 9p21 associated ALS/FTLD populations, suggests it is the most important genetic factor contributing to disease in the center of the disease spectrum linking ALS and FTLD (table 1). Moreover, next to the chr9p21 conclusively linked ALS-FTLD families, several other (smaller) families were also reported without conclusive linkage but with several indications pointing towards the presence of a segregating haplotype in the ALSFTD2 locus (Krueger et al., 2009; Le Ber et al., 2009; Momeni et al., 2006; Pearson et al., 2011; Valdmanis et al., 2007; Yan et al., 2008) (table 1). Identification of this major gene will undoubtedly be a steppingstone for subsequent cell biological studies aiming at better understanding of the pathobiology of neurodegenerative processes leading to ALS and FTLD.
