The present study reports the results of a large and comprehensive genome-wide screening of the genetics of gene expression in an attempt to find novel genetic variants that associate with sporadic ALS. We used a two-stage approach to minimize the chance of false-positive findings, both for eQTL discovery purposes and for the detection of novel SNP-ALS associations. eQTLs were used for prioritizing GWAS results, as it has been established that SNPs that are truly associated with disease are more likely to be eQTLs 
. In the present study, we show that the number of eQTLs is greater than expected by chance (p
0.003) among the SNPs with a nominal association with ALS, compared to frequency-matched SNPs, also indicating that eQTLs may be useful in the prioritization of GWAS results in ALS. We identified eight SNPs in one cis
eQTL, modulating CYP27A1
gene expression levels, which replicated in the second eQTL dataset and second GWAS set. The eQTL SNPs within this locus are part of a large linkage disequilibrium (LD) block comprising a total of ten genes (). The figure clearly shows that the strongest eQTL associations exist for SNPs modulating CYP27A1
expression, explaining up to 65% of variation in gene expression of this gene. Additionally, we show that C9orf72
appears to be the only gene in the 9p21.2 locus that is regulated in cis,
showing the potential of this approach in identifying causative genes in association loci in ALS.
Regional linkage disequilibrium (LD) near the CYP27A1 locus on chromosome 2.
As shown in Table S3
, the SNPs modulating transcript levels had small effect sizes in our joint GWAS association results, the highest odds ratio (OR) being 1.13. We used PS v3.0 
for statistical power calculations to determine the required sample size for a third genotypic replication of such SNPs. In order to replicate an association for one SNP with minor allele frequency 0.35 at α
0.05, one would require a minimum of 2,250 cases and 2,250 controls to achieve 80% power for detecting an effect with OR 1.13. As shown in , several eQTL SNPs did not reach Bonferroni corrected significance in the replication data alone, but do show stronger effects in the joint GWAS data, indicating that statistical power of the GWAS replication set might be a limiting factor. By testing these SNPs in a third independent replication cohort, additional true associations may be detected. The required sample size for such an effort would, however, increase dramatically when adding more tests. Further international collaboration, therefore, is needed in order to achieve sufficient statistical power for the replication of SNPs with small effect sizes.
We searched MEDLINE, Gene Ontology and OMIM databases to identify links to known pathways in ALS pathogenesis for CYP27A1
. The CYP27A1
gene is involved in cholesterol metabolism and has been associated with cerebrotendinous xanthomatosis (CTX), which can present with progressive upper motor neuron signs and is a known clinical mimic for primary lateral sclerosis 
. Two heterozygous mutations in CYP27A1
have been reported in a patient with atypical CTX and frontotemporal dementia characteristics 
. Furthermore, previously, serum cholesterol levels have been implicated in modifying survival and in the onset of respiratory impairment in ALS patients 
. The combination of our results and these prior data make CYP27A1
a plausible candidate gene for ALS.
The strengths of our study are the meticulous pruning of expression probes as present on the expression array, with regard to non-specific mapping in the human transcriptome, or harboring SNPs that might interfere with hybridization of probes to the array, resulting in false-positive eQTLs 
. In addition, permutation schemes were applied, preserving the LD structure within subjects, also minimizing the detection of false-positive eQTLs. Finally, a two-stage approach, both for eQTLs discovery purposes and for the detection of novel SNP-ALS associations, ensures robustness of the results.
A drawback of the present study lies in the use of whole blood instead of neuronal tissue for the measurement of mRNA expression levels. As neuronal tissue is inaccessible in living ALS patients, one could consider the use of human neuronal tissue from autopsy. However, in post-mortem material of ALS patients, most affected motor neurons will have degenerated and one would be investigating exclusively end-stage disease expression profiles. We have investigated the proportion of overlapping eQTLs between our study and other studies, including two studies on human brain tissue (Table S4
. Studies of the genetics of gene expression appear to have modest overlap in the eQTLs identified. For example, 36.1% of genes mapped by a cis
eQTL in lymphocytes were identified in a study using lymphoblastoid cell lines 
. A smaller overlap (22%) was found between two studies on brain tissue, which may partly be due to low statistical power 
. In the present study, 37 – 52% of the genes mapped by cis
eQTLs in human brain tissue studies appeared to be present in our data (Table S4
). The proportion of overlap with studies on blood-derived tissues was comparable (41 – 45%). Considering the relatively high concordance of genes mapped by cis
eQTLs in our screen with those found in human brain tissue, we consider blood to be a valid starting point for genetic mapping of gene expression in ALS. A large collection of central nervous system tissue control samples may, however, further boost the discovery of novel genetic variants that are associated with ALS.
The focused analysis of variants in the chromosome 9p21.2 locus, which was previously associated with ALS 
, did not identify rs2814707 or rs3849942 as eQTL SNPs. We did, however, find evidence of two other SNPs (rs10122902 and rs1565948), located within a large LD block surrounding the previously associated markers, to be correlated with altered expression levels of C9orf72 isoform a
. SNP rs1565948 was associated with ALS in our joint GWAS data. The rs10122902 variant was not associated with ALS in our joint GWAS, but was previously shown to be part of a haplotype with rs3849942, in which the major allele of rs10122902 was associated with increased risk of ALS 
. Genetic variation in the chromosome 9p21.2 locus, therefore, appears to be associated with altered gene expression of C9orf72
. The recent discovery of the intronic hexanucleotide repeat expansion in C9orf72
on a common haplotype in 9p21.2 linked families with ALS and FTD 
thus illustrates the potential of the combined use of gene expression and genotyping in search for causative genes in human diseases. The mechanism though of the recently discovered repeat expansion in C9orf72
remains to be established. There could be a direct effect of expression levels of isoforms of C9orf72
, or a “trans”-like effect through RNA-toxicity, as shown in other repeat expansions diseases including fragile X-associated tremor/ataxia syndrome (FXTAS) 
. Other types of experiments are needed to elucidate this mechanism.
In summary, our genome-wide study of the genetics of gene expression has identified one cis
eQTL for sporadic ALS, which modulates CYP27A1
expression and additionally points to C9orf72
in the chromosome 9p21.2 locus as the gene involved in ALS pathogenesis. To further identify eQTLs relevant to ALS, the concomitant analysis of epigenetic and other level -omic data, e.g. proteomic or metabonomic can be used, as recently shown in a model organism 
. These studies are preferably performed in ‘ALS target tissues’, including post-mortem central nervous system tissues and induced pluripotent stem cells differentiated to a neuronal or glial lineage. Such studies may provide us with more insight into novel pathogenic pathways and networks causal to this devastating disease.