Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) with presumed autoimmune origin, triggered by genetic and environmental risk factors. The aetiology of MS is unknown, and the pathology is not well understood. In addition to those SNPs identified as significant in a 2009 GWAS (Baranzini, Wang et al. 2009) we investigated two SNPs in enzymes responsible for the initiation and modification of the side chains characteristic of HSPGs (EXT1, SULF1) and another HSPG core protein, SDC1. No significant difference was observed in our MS population in relation to the EXT1 and SULF1 SNPs examined. Further analysis revealed no association with disease subtype. However, in this study we did identify significant associations with SDC1, GPC5 and GPC6 polymorphisms. Overall, analysis by disease subtype maintained this significance, as did analysis by sex.
While this study may suggest that no association exists between EXT1 and MS, previous studies have indicated strong expression of EXT1 in the developing brain (Inatani and Yamaguchi 2003). Additionally, it has been suggested that EXT1 correlates with the sites of active neuron generation (Inatani and Yamaguchi 2003). Prenatally EXT1 has been localised in the neuroepithelial cells, which surround the lateral ventricles, cerebral cortex and hippocampus. However, in the postnatal stage EXT1 is expressed in the cerebellum, which may correlate with the symptoms seen in MS such as ataxia (Inatani and Yamaguchi 2003). In a murine model complete abrogation of EXT1 results in embryonic lethality during gastrulation due to the absence of heparan sulfate (Holst, Bou-Reslan et al. 2007). EXT1 alone is able to polymerise GAG chains for attachment to PG core proteins (Busse and Kusche-Gullberg 2003, Kim, Kitagawa et al. 2003), however, both EXT1 and EXT2 are required for in vivo HS chain elongation (Busse-Wicher, Wicher et al. 2014).
SULF1 has been suggested to have a role in the brain, however deficiencies in SULF1 have been associated with developmental abnormalities such as decreased body mass and subtle kidney and bone defects (Holst, Bou-Reslan et al. 2007). SULF1 has also been linked to tumour suppressor functions as it has been reported to be downregulated in some cancers (Han, Huang et al. 2011).
HS chain biosynthesis is a complex process requiring the action of enzymes such as SULF1 to generate their complex sulfation patterns through addition and removal of sulfation. Successful binding of growth factors to GAG chains for signalling pathway activation requires specific sulfation patterns on these side chains. The interaction between HS and FGF-2 is a well-documented example where HS requires 2-O sulfation to be able to bind to FGF-2. Without this binding, cellular proliferation and differentiation are inhibited. Although, these two SNPs in EXT1 and SULF1 showed no significance in our population of moderate size, further investigation should be undertaken with a larger cohort before excluding the possibility of their involvement in MS susceptibility.
In the first of the HSPG core proteins examined (SDC1) we found a significant association between the SNP, rs1131351, and MS. This association revealed a stronger link between the SNP and females suffering from early onset forms of disease (PPMS, RRMS). SPMS occurs 8–20 years after RRMS onset (Trapp and Nave 2008) with the negative association seen here with this disease state reflecting a role for SDC1 in the initiation of disease. Females with PPMS and the minor allele of SDC1 have more than double the risk (OR = 2.24) of developing MS than controls. In patients suffering from RRMS this increase in risk is approximately 1.5 times (OR = 1.57). This could be due to the fact that PPMS seems to be more aggressive during onset when compared with RRMS. Even though they are both classified as onset stages of the disease, RRMS can progress to SPMS, with reversible damage occurring in this stage, while PPMS damage is irreversible and the symptoms are generally more detrimental (reviewed in (Goldenberg 2012, Dutta and Trapp 2014).
Active MS lesions are characterised by an influx of inflammatory cells and a decrease of chondroitin sulfate proteoglycans (van Horssen, Bo et al. 2006). Furthermore, white matter-associated proteoglycans have been known to accumulate in macrophages, suggesting that chondroitin sulfate proteoglycans are phagocytosed with myelin or their breakdown products (van Horssen, Bo et al. 2006). SDC1 contains ser-gly sequences that may serve as an attachment site for chondroitin sulfate (Bernfield, Gotte et al. 1999) while also carrying HS chains. Therefore, a mutation in SDC1 may result in activation of the macrophages causing phagocytosis, consequently leading to a reduction in SDC1 in MS patients. In addition, TGF-β along with FGF-2, have been linked to enhanced expression of SDC1 (Bernfield, Gotte et al. 1999). Enhanced expression of TGF-β has been observed in MS lesions causing matrix deposition by the promotion of transcription genes and suppression of degrading enzymes (van Horssen, Bo et al. 2006). FGF-2 has been associated with the survival, proliferation and migration of oligodendrocyte precursors leading to the promotion of remyelination (van Horssen, Dijkstra et al. 2007). This contradicts the mechanism of neurodegeneration seen in MS patients however, FGF-2 may be a survival mechanism established to reverse the damage particularly in relapsing and remitting MS patients, as it has binding partners other than SDC1.
In addition to the SDC1-FGF-2/TGF- β signalling mechanisms, TNF-α has been demonstrated to decrease SDC1 expression in cultured endothelial cells (Bernfield, Gotte et al. 1999). TNF-α has been shown to be involved in the inflammatory response (Titelbaum, Degenhardt et al. 2005) and could be involved in the process mimicking the early stages of MS where breakdown of the blood brain barrier allows inflammatory cells to cross into the brain and contribute to demyelination and axonal damage (van Horssen, Bo et al. 2006).
In this study we aimed to replicate and build on results from a number of previous GWAS and replication studies in an Australian case-control population. These earlier results implicated GPC5 and GPC6 SNPs in MS. Our analysis of three GPC5 and two GPC6 SNPs also identified significant associations between these genes and MS. GPC5-rs10492503 showed a significant association in the total disease population. When analysed further we found significant associations with two disease states (SPMS and RRMS) and in the female population and the female SPMS and RRMS subtypes. GPC6-rs17267815 showed a minor significant association within the RRMS subtype only. Further analysis suggested this association was due to the male RRMS subgroup, however due to low sample numbers once the population was stratified, significance values are suggestive only.
We identified no LD between the SNPs studied within the previously identified 13q31-32 risk region containing both these genes, nor could we replicate the moderate LD identified previously in GPC5 (Lorentzen, Melum et al. 2010). All five GPC5 and GPC6 SNPs investigated in this study had previously been identified as significant in large-scale case/control GWAS and replication studies in Norwegian and Spanish populations (Comabella, Craig et al. 2008, Baranzini, Wang et al. 2009, Cenit, Blanco-Kelly et al. 2009, Lorentzen, Melum et al. 2010) with varying and often contradictory levels of significance. SNPs reaching significance in one study were not found to be significant in another (Baranzini, Wang et al. 2009, Lorentzen, Melum et al. 2010, Cavanillas, Fernandez et al. 2011). Analysis by disease state of some of these populations determined significant associations with the RRMS subtype (Poliseno, Salmena et al. 2010). Indeed, in our population, when a significant association was observed in these genes, it was often significant in the RRMS sub-population. This may be due to the mixture of the populations as patients from pure Northern European ancestry have a higher risk of developing MS (Kurtzke, Beebe et al. 1979). While our Australian population is of Caucasian decent, it is not necessarily of purely Northern European origin, explaining some differences between results and levels of significance identified in these studies. In addition, while our results are not strongly significant on their own, they replicate previous studies and support and strengthen the evidence for involvement of GPC5 and GPC6 in the development and progression of MS.
Many HSPGs and their associated enzymes have been associated with disease, with both SDC1 and SDC4 showing strong involvement with breast cancer (Tkachenko, Rhodes et al. 2005, Lendorf, Manon-Jensen et al. 2011, Okolicsanyi, van Wijnen et al. 2014). As yet, the functions of the glypicans physiologically, in both normal and pathological conditions, remain poorly understood. However, data here and in other studies suggest an important function for these proteins in cell growth and regulation of division. Celie and colleagues suggested that HSPGs are involved in the inflammatory response and have a regulatory role in leukocyte extravasation (Celie, Beelen et al. 2009), a condition synonymous with MS. Glypicans have been shown to play roles in diseases such as hepatocellular carcinoma (GPC3; (Capurro, Xiang et al. 2005)) and Simpson-Golabi-Behmel syndrome (GPC3/GPC4; (Veugelers, De Cat et al. 1999)). While the function of GPC5 remains poorly understood, especially in MS, different polymorphisms have been reported to increase the risk of lung cancer in non-smokers (Li, Sheu et al. 2010) while decreasing the risk of cancer in MS patients, with this reduced cancer risk stemming from the glypican gene (Handel and Ramagopalan 2010). In addition, the gene region 13q31-32 containing both GPC5 and GPC6 has also previously been associated with increased risk of Primary Sclerosing Cholangitis (PSC), a chronic liver disease where a strong association has been identified between the SNP GPC6-rs9524260 and disease (Karlsen, Franke et al. 2010).
Due to the interaction of glypicans with several growth factors, chemokines and extracellular matrix proteins, this may affect neural growth and repair (Byun, Caillier et al. 2008). The results of a study by Cenit and colleagues (2009) not only supported a significant association of GPC5-rs10492503 with MS, but also indicated approximately twice the risk of developing disease in an individual who has one or more copies of the variant allele (Cenit, Blanco-Kelly et al. 2009). GPC5 has been reported to play an important role during the process of cell division and growth regulation. It is predominantly expressed in foetal tissues, including brain, lung, liver and kidney. However, it has an exclusive expression in adult tissue in the CNS and in its neurons (Saunders, Paine-Saunders et al. 1997, Veugelers, De Cat et al. 1999). This suggests a possible and plausible role for this gene in controlling various neurotropic factors and maintenance of neural function. In our study we found a significant association of this GPC5 variation with the early onset from of disease (RRMS) and also the severe form (SPMS), which is a progression of disease and characterised by irreversible damage suggesting a role for GPC5 in the progression of MS. GPC5 plays an important role in brain patterning, synapse formation, axon regeneration and guidance. Its expression in the in the developing brain and the adult CNS (the origin of MS) also support a role for this gene in different disease states.
In PPMS most of the myelin degradation occurs in the cerebrum and cerebellar cortex of the CNS (Kutzelnigg, Lucchinetti et al. 2005). Dysfunction of GPC5 would affect cell proliferation and tissue growth. With the cells no longer able to interact with the various positively charged growth factors, this would affect brain patterning, synapse formation, and an interruption in axon regeneration. This suggests, that abnormal GPC5 may trigger MS and the subsequent disability experienced by sufferers.
Further evidence supporting this hypothesis is data demonstrating that HSPGs have been identified in the active lesions of MS, where they are thought to be involved in the sequestering of pro-inflammatory chemokines (van Horssen, Bo et al. 2006). GPC5’s expression and interaction with various growth factors and chemokines likely affects growth and repair of neurons, also influencing the guidance of axons and synapse formation (Lee and Chien 2004, van Horssen, Bo et al. 2006, Van Vactor, Wall et al. 2006). Indeed, another member of the glypican family (GPC1) has been shown to be required for Schwann cell myelination (Chernousov, Rothblum et al. 2006). With documented involvement of other glypicans, it is plausible allelic variants of GPC5 may affect neuronal repair, axon guidance and new synaptic formation.
The embryonic expression of GPC6 is detected in the ovary, liver and kidneys, while in the adult, it is detected only in the ovary and intestine (Fransson 2003). A significant role for this HSPG gene has been implied in Neuroticism. This is a moderately heritable personality trait considered to be a risk factor for developing major depression, anxiety disorders and dementia (Calboli, Tozzi et al. 2010). This may indicate a role of the gene in neural diseases with origin in the CNS, the location of the MS associated lesions. Overall, the functional role for GPC6 is poorly understood, but this study provides evidence of a role for GPC6 in MS.