Identification of genes predisposing to MS has proven to be a daunting task. A large number of genome-wide linkage scans have been conducted, but the strong linkage and association with the HLA
locus remained to be the only systematically replicated finding with a genome-wide significance until 2007 (8
). Finally, the first MS GWA study (10
), together with other studies (11
), revealed two additional loci, IL7R
, associated with an increased risk of MS. These loci have now been replicated by independent groups of investigators (30
) and are associated with MS susceptibility at a genome-wide level of significance. Despite of initial disappointment often faced with HLA-associated diseases, we are beginning to uncover the genetic architecture of this disease. However, the risk alleles in the IL7R
are estimated to explain only a small proportion (0.2%) of the genetic risk of MS (10
). Many more such low penetrance alleles remain to be discovered using conventional GWA approaches, but a change of tack will also be needed to discover relatively rare alleles, enriched in some populations or familial study samples, and potentially having a stronger effect on disease susceptibility. Here, we have attempted to pinpoint a rare MS predisposing gene on the 5p linkage region, for which most of the linkage information emerges from an internal founder population. An effort was previously made to narrow the wide locus (34
), but only the most ‘telomeric’ 20 cM of the original wide 40 cM-linked region was analyzed in the restriction step, as the LODs were highest (maximum LOD 3.4) in that area. Importantly, no gene in the restricted region was found to explain the linkage observed (data not shown). Here, the complete ~40 cM-linked region was scanned using the Illumina HumanHap300 panel.
In this linkage-guided association study, we initially used the haplotype association analysis results for ranking the most interesting regions for follow-up. Scanning of the complete linkage region of 45 Mb revealed strong association only with one haplotype flanking the C7 and FLJ40243 genes, and this finding was validated in an independent case–control set originating from the same high-risk geographical region. The same markers also provided nominal evidence for association in a family based analysis. The association analysis combining all the samples from the high-risk region resulted in a P-value of 3.2 × 10−6, which remained significant even after the conservative Bonferroni correction.
Eighty-nine percentage of the MS cases used in the initial screen had both parents born within the MS high-risk region, and genealogical studies showed that majority of the patients were distantly related. Thus, our study design was specifically aimed to enrich for relatively rare, potentially more penetrant variants. Indeed, the identified risk allele was found to be present only in ~4% of alleles in the general European population, whereas it has a slightly higher frequency of ~6% in the Asian populations and it is almost absent among Africans, Southern Americans and Oceanians. Importantly, it has obviously become enriched in the Southern Ostrobothnian high-risk MS region due to the founder effect and isolation, and is found in 12% of cases. Within the high-risk region, carriers of the C7-FLJ40243
risk allele have higher risk to get the disease compared with non-carriers (OR: 2.7), whereas the previously identified MS risk alleles in IL7R and IL2RA have been reported to have OR of 1.2 (32
). Suggestive evidence for association with C7-FLJ40243
alleles and MS was seen also in more heterogeneous populations, but as expected, there seems to be more allelic heterogeneity within the region, and the causative variant is most probably carried in diverse allelic backgrounds.
The advantages of the Finnish population and, especially, its subisolates are that most of the affected individuals typically share the same major risk allele and that the relatively rare variants can be exposed by the common HapMap markers due to the wide LD intervals (35
). However, it is worth noting that these relatively rare variants identified using population isolates are much more challenging to detect with common HapMap SNPs in more heterogeneous populations having distinct LD patterns. Moreover, it is also possible that there are various causative mutations within the same gene in different populations. Thus, replication of the original allelic association in a more heterogeneous population might not be straightforward. This holds true, for example, for familial hypercholesterolemia [FH (MIM #143890)], an autosomal dominant disorder, where majority of mutations occur in the gene encoding low-density lipoprotein receptor (LDLR
) and in two other genes (36
). There is notable allelic heterogeneity in FH patients worldwide. Over 1000 mutations have been reported (37
), and allelic heterogeneity is seen even within Finland in LDLR
: there is a founder mutation (FH-North Karelia) found as the major mutation in a limited region in the Northeastern part of Finland and other mutations are found elsewhere in Finland (38
has previously been studied as an MS candidate gene (39
). Three SNPs, C7-327 corresponding to rs7713884 in intron 13, C7-363 corresponding to rs13157656 in exon 14 and a SNP called C7-396, were genotyped in 227 MS families (including only 59 whole trio families) and 72 controls originating from UK. Suggestive evidence for linkage was observed (P
= 0.0061). Further, SNP rs7713884 provided nominal evidence for association in the case–control analysis (P
= 0.009), but family based association with the transmission disequilibrium test (TDT) was not seen with any of the SNPs, and the authors concluded that C7
does not confer susceptibility to MS. Notably the family based study was underpowered.
Both human C7
(C7) and FLJ40243
(RIKEN cDNA 4930455B06 gene) exist also in the corresponding location of the mouse chromosome 15 (Mouse Genome Informatics database, http://www.informatics.jax.org/
). The genes coding for the sixth and ninth components of the complement cascade [C6
(MIM +217050), C9
(MIM +120940)] are also located in the chromosome 5-linked region, but no signal of association with MS was seen with those in the Finnish GWA study sample.
Based on our results, neither C7 nor FLJ40243 was expressed in PBMCs. Thus, expression levels of these genes could not be measured in RNA samples obtained from mononuclear cells of MS patients. However, we tested expression of FLJ40243 in human tissue cDNA panels. FLJ40243 was observed to be expressed in spleen, lymph node, fetal liver and fetal skeletal muscle, but no evidence for expression was seen in fetal brain, adult thymus, tonsil, bone marrow or peripheral blood leukocytes.
C7 is synthesized not only by hepatocytes, but also by endothelial cells, polymorphonuclear cells, macrophages, platelets, fibroblasts, synovial tissue and even in the CNS by astrocytes and oligodendrocytes (40
), cells relevant for MS. We measured circulating C7 protein levels and total complement activity in MS patients and in unaffected controls. The causative variant of the C7-FLJ40243
risk region is yet unknown, but based on our results, it may affect protein levels of the complement component 7, which is an excellent functional candidate for MS. Importantly, the complement system was observed to be significantly more active among MS cases compared with unaffected population controls; the MS patients carrying the identified C7-FLJ40243
risk haplotype producing most functional TCCs when the complement cascade gets activated.
The complement system is a biochemical cascade of the innate immune system that helps to clear pathogens from the organism. In addition, the first components (C1–C4) of the classical and the lectin pathway are important in the clearance of apoptotic cells and cellular debris (45
). Activation of C3 by the classical, lectin or alternative pathway leads to activation of the terminal components (C5–C9), which then form the TCC (C5b–C9), also called as the MAC when assembled on a cell membrane. The MAC forms a transmembrane channel on the cell membrane of the target cell, resulting in the osmotic lysis of the target. The classical complement pathway requires antibodies for activation, whereas the alternative pathway can be activated directly by microbial surfaces and the lectin pathway by certain carbohydrates, such as mannan. The end result of each pathway is in any case the formation of the MAC.
C7 is a single-chain plasma glycoprotein involved in the cytolytic phase of the complement activation cascade. C7
deficient individuals (MIM #610102) are prone to Neisseria meningitidis infections, because the MAC complex, which is important in destroying gram-negative bacteria by cytolysis, cannot be formed correctly. Importantly, C7 is a critical limiting factor of complement activation: only when the local expression of C7 is sufficient, C7 binds to preformed C5b6 and the resulting C5b–C7 complex is able to insert into the phospholipid membrane to start the formation of the MAC (46
). The oligodendrocytes are, especially, sensitive to complement-mediated injury because of the relative deficiency of protective complement regulatory proteins, which normally protect host cells from complement-mediated lysis (47
Several publications support the hypothesis that complement activation and especially the MAC may play a key role in the pathogenesis of autoimmune demyelination. Increased levels of terminal complement complexes (C5b–9) have been detected in the cerebrospinal fluid of MS patients during relapses, and their levels have been shown to correlate with the Expanded Standard Disability Status Scale score, measuring the neurological disability (48
). C6-deficient rats, unable to form the MAC, exhibit no demyelination and show a significantly reduced clinical score in the Ab-mediated demyelinating form of EAE, the animal model of MS (49
). Moreover, C9 deposition (corresponding to the number of MAC complexes), P-selectin expression and cellular infiltrates were observed to be reduced in the spinal cords of these rats compared with rats capable of forming the MAC (50
). On the other hand, mice lacking the complement regulatory protein CD59, which normally protects host cells from complement-mediated damage by blocking the formation of the MAC, have more severe EAE and increased demyelination, inflammatory cell infiltration and axonal injury (51
It is tempting to speculate that the causative variant of the C7-FLJ40243
region may lead to increased complement activation in the CNS and further to demyelination by damaging the defenceless myelin forming oligodendrocytes. MS can be classified into four different pathological patterns based on the composition of active lesions (52
). Pattern II, accounting for over 50% of patients, is characterized by immunoglobulins and MAC deposition at the areas of myelin destruction. Thus, the C7-FLJ40243
risk allele identified here may predispose, especially, to this type of disease.
To summarize, the C7-FLJ40243 risk region, here shown to contain a risk allele enriched in a population isolate, is an excellent candidate for the susceptibility of autoimmune demyelination and seems to be a second MS locus in this region on 5p, initially identified by linkage in multiple study samples. Our observations highlight the value of population isolates in the identification of rare disease alleles in common diseases and further demonstrate the complexity of the genome regions initially identified as potential risk loci.