INTRODUCTION
============

Multiple sclerosis (MS) is a chronic demyelinating and inflammatory disease of the the central nervous system (CNS).[@B1][@B2] Genetic factors contribute markedly to the susceptibility to MS, since the children of affected parents have a tenfold higher risk of developing the condition than the general population.[@B3]

It has been demonstrated that the human leukocyte antigen (HLA) gene is closely related to MS susceptibility. The HLA gene contains a great many genes (HLA class I, II, and III) residing on chromosome 6, which is related to immune system function in humans. The proteins encoded by the HLA class I and II regions are involved in antigen processing and presentation, and play a major role in autoimmune events. MS is believed to be an immune-mediated disorder that leads to recurrent immune attacks on the CNS.[@B4]

Recent genome-wide association studies (GWASs) have identified many loci with modest effects.[@B5][@B6][@B7] However, previous GWASs used single-nucleotide polymorphisms (SNPs) as a basic analysis unit,[@B5][@B6][@B8][@B9] and adopted stringent thresholds of significance to control for the false-positive rate. This approach resulted in a large number of SNPs with potential effects being filtered out and ignored.

The gene-based GWAS study strategy, involving analyzing all variants within a putative gene, has proved to be more powerful than regular single-SNP-based GWASs for detecting disease susceptibility genes.[@B10][@B11][@B12] To detect \"novel\" genes associated with MS, we performed a gene-based GWAS using the Knowledge-based mining system for Genome-wide Genetic studies (KGG; <http://statgenpro.psychiatry.hku.hk/limx/kgg/index.html>)[@B10] in an initial study sample containing 931 family trios.[@B8] We also performed other functional analyses to supplement the evidence regarding the relevance of the novel genes to MS.

METHODS
=======

Samples
-------

The initial GWAS sample included a total of 931 family trios, each of which consisted of an affected MS child and both parents. The replication sample contained 978 cases and 883 controls. The MS patient was diagnosed according to the McDonald criteria.[@B13] Both the initial and replication study samples included all clinical subtypes and partial clinically isolated syndromes. However, the replication study had the priority to include patients with the relapsing onset form of MS. Institutional Review Board was exempted because this study used public available database of Genotypes and Phenotypes (dbGaP) which dose not involve any personal information. The study samples, genotyping, quality control, and SNP exclusion criteria have been described previously.[@B8][@B9]

Gene-based GWAS
---------------

The gene-based GWAS and replication analysis were based on the probability values generated in previous genome-wide SNP-based association studies and downloaded from the dbGaP (<http://www.ncbi.nlm.nih.gov/gap/?term=multiple+sclerosis>, accession number: phs000139 and phs000171). We used the Gene-Based Association Test Using Extended Simes Procedure analysis method modeled in KGG 2.5 (<http://statgenpro.psychiatry.hku.hk/limx/kgg/index.html>).[@B10] The SNPs, ranging from the upstream 5 kb at the 5\' end to the downstream 5 kb at the 3\' end, were assigned to one gene. In total, 48% of SNPs across the whole genome were assigned to genes.

Differential expression analysis for MS-associated genes
--------------------------------------------------------

We downloaded four gene-expression data sets from the Gene Expression Omnibus (GEO) of the National Center for Biotechnology Information (<http://www.ncbi.nlm.nih.gov/geo>; GSE21942, GSE27694, GSE16461, and GSE52139). A case-control study design was used to investigate all four data sets, and multiple cell types were investigated, including peripheral blood mononuclear cells (PBMCs), CD34+ hematopoietic progenitor cells (HPCs), CD8+ T lymphocytes, and spinal cord. The study design data analysis are described in detail elsewhere.[@B14][@B15][@B16][@B17] Differentially expressed genes between MS cases and controls were identified by comparing mean gene-expression signals in MS cases versus controls and analyzing the findings using *t*-tests.

Protein-protein interaction network
-----------------------------------

MS-associated gene interactions and associations were detected by protein-protein interaction analysis, conducted by searching the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database (<http://string-db.org/>). The STRING database integrates known and predicted associations derived from a genomic context, high-throughput experiments, coexpression, and previous knowledge (text mining).[@B18]

Functional annotation clustering analysis
-----------------------------------------

The probability of the identified MS-associated genes clustering in a Gene Ontology (GO) term or a particular biological pathway as defined by the GO project and Kyoto Encyclopedia of Genes and Genomes database was tested by performing a functional annotation clustering analysis using the Database for Annotation, Visualization and Integrated Discovery integrated database query tools (<http://david.abcc.ncifcrf.gov/>).[@B19][@B20] The enrichment was measured quantitatively using Fisher\'s exact test, and the Bonferroni method[@B21] was adopted to correct for multiple testing.

RESULTS
=======

Quantile-quantile plots of the association results for the original genome scan and the present gene-based GWAS are shown in [Fig. 1](#F1){ref-type="fig"}, in which probability values (shown as -log10 values) for all 20,761 genes and all SNPs are plotted against the expected null distribution. The tail of the distribution of gene-based probability values deviated more significantly than those of SNPs inside or outside of the gene. The distribution of the probability values for the present gene-based GWAS is shown in [Fig. 2](#F2){ref-type="fig"}.

Based on a false discovery rate threshold of 1.40×10^-4^, 58 genes were found to be significantly associated with MS in the initial gene-based GWAS ([Supplementary Table 1](#S1){ref-type="supplementary-material"} in the online-only Data Supplement). Among those 58 genes, 53 were located in the HLA region on chromosome 6. In contrast, according to the raw SNP-based probability values, 75 SNPs in 31 genes were found to be significantly associated with MS (threshold of *p*\<1.49×10^-7^). Therefore, the present gene-based GWAS detected 27 novel genes that had not previously been detected in the original SNP-based GWAS. After searching the Phenotype-Genotype Integrator ([www.ncbi.nlm.nih.gov/gap/phegeni/](http://www.ncbi.nlm.nih.gov/gap/phegeni/)), a database that archives previous association results, we found that 7 of the 27 genes had already been reported for significant associations (threshold of *p*\<5.0×10^-5^). Therefore, among the 58 MS-associated genes that we identified, 20 were considered as novel MS candidate genes. Of note, five of these novel genes were located outside the HLA region.

Among the total 58 identified MS-associated genes, 44 genes with genotype data in the replication sample were subjected to further association tests, which revealed that 35 of them (80%) were still significantly associated with MS (*p*\<0.05). Furthermore, 21 of these 58 genes, including 8 novel genes, exhibited differential expression in the differential expression analysis ([Table 1](#T1){ref-type="table"}). Most interestingly, the genes encoding histone-lysine N-methyltransferase, H3 lysine-9 specific 3 (*EHMT2*), major histocompatibility complex, Class I, A (*HLA-C*), and negative elongation factor E (*RDBP*) exhibited significantly differential expressions between cases and controls in both PBMCs and CD34+ HPCs, and the gene encoding myelin oligodendrocyte glycoprotein (*MOG*) in PBMCs, CD8+ T lymphocytes, and spinal cord, simultaneously ([Table 1](#T1){ref-type="table"}).

The 58 identified MS-associated genes were retrieved from the STRING database. Only 36 genes, including 10 novel genes, were annotated in this database. The genes at the HLA regions were clearly enriched into two clusters: HLA class I and II clusters ([Fig. 3](#F3){ref-type="fig"}). Two novel genes, those encoding major histocompatibility complex, class, I, A (*HLA-A*) and major histocompatibility complex, class II, DM alpha (*HLA-DMA*), were involved in HLA-class I and II clusters, respectively. Another four novel genes, *MOG*, and those encoding coiled-coil alpha-helical rod protein 1 (*CCHCR1*), bromodomain-containing protein 2 (*BRD2*), and chromosome 6 open reading frame 15 (*C6orf15*), were directly connected with the HLA clusters ([Fig. 3](#F3){ref-type="fig"}).

Taking both the replication data and gene-expression data together, we found that 15 novel genes were supported by replicated association and/or differential expression ([Table 2](#T2){ref-type="table"}). Most interestingly, the significance of four genes---*HLA-DMA*, *CCHCR1*, *MOG*, and the gene encoding HLA complex group 22 (*HCG22*)---in MS was supported by both the replication and gene-expression studies. In addition, the significance of two non-HLA genes---those encoding family with sequence similarity 69, member A (*FAM69A*) and POU domain class 2 transcription factor 3 (*POU2F3*)---in MS was supported by evidence of differential expressions in PBMCs and CD8+ T lymphocytes, respectively.

The identified 58 MS-associated genes were found enriched in 54 GO terms, even after Bonferroni correction (*p*\<0.05) ([Supplementary Table 2](#S2){ref-type="supplementary-material"} in the online-only Data Supplement). Most significantly, 12 genes were enriched in \"antigen processing and presentation\" (GO: 0019882; *p*=271.09×10^-16^), and 11 genes were enriched in \"MHC protein complex\" (GO: 0012611; *p*=1.06×10^-16^).

DISCUSSION
==========

Gene-based association analysis is an efficient method for detecting associations between candidate genes and complex diseases, as it combines signals for all variants within a putative gene. By using this method, several studies have identified new associations between genes and diseases.[@B22][@B23] The present study again highlights the superior power of gene-based association analysis for detecting associations for MS. Specifically, we detected 58 genes significantly associated with MS, including 20 novel genes that were undetected in previous single SNP-based GWASs. Furthermore, associations for 80% of the identified MS-associated genes were replicated.

Most previous association studies have identified only the statistical relevance of genes to MS (at the DNA level), without dissecting the functional mechanisms underlying those associations. In contrast, in the present study we not only established statistical associations between genetic markers and MS at the DNA level, but also performed follow-up differential gene expression analyses and functional annotation clustering analyses as important supplementary methods to analyze the function of causal variants. This supplementary evidence strengthens the likelihood that the eight novel genes identified in this study with significantly differential expressions are directly involved in the pathogenesis of MS.

Recent GWASs have identified a great number of MS-associated genetic loci,[@B5][@B9][@B24][@B25][@B26] most of which have been mapped to the HLA region. Most of the loci identified in the present study were also located in this region. As we know, the major HLAs are essential elements for immune function. Therefore, our findings also highlight the importance of the autoimmune system in the etiology of MS.

Most interestingly, evidence from association, replication, and differential expression studies strongly supports the significance of the following four genes to MS: *MOG*, *HLA-DMA*, *CCHCR1*, and *HCG22*. Up to now, the biological function of *HCG22* in MS or immunity is unknown. *HLA-DMA* and *CCHCR1* exhibit suggestive associations with rheumatoid arthritis (RA) and psoriasis,[@B27][@B28][@B29] respectively, suggesting that both genes are involved in the immune response. The protein encoded by *MOG*, myelin oligodendrocyte glycoprotein (MOG) is a membrane protein expressed on the surface of oligodendrocyte cells and myelin sheaths.[@B30] MOG is an important candidate target antigen in MS.[@B31][@B32][@B33] Monoclonal antibodies against MOG were used to develop an animal model of MS.[@B34] The functions of these genes in MS need further investigation.

In addition, three genes (*POU2F3*, *BRD2*, and *FAM69A*) were expressed differentially in MS-related cells. *POU2F3*, a non-HLA gene (chromosome 11), is associated with melanoma and cervical cancer.[@B35][@B36] *BRD2* is associated with cancer, obesity, type 2 diabetes, RA, and inflammation,[@B37][@B38][@B39] and was differentially expressed in the PBMCs of patients with RA (*p*=2.31×10^-3^, data sets from GEO; [www.ncbi.nlm.nih.gov/geo](http://www.ncbi.nlm.nih.gov/geo), GSE\#: GSE15573). The associations with other autoimmune diseases suggested the possible relevance of *BRD2* and *POU2F3* to MS. The specific functions of *FAM69A* in MS are unknown. A previous study found that 21 SNPs located at the GFI-EVI5-RPL5-FAM69A locus were positively associated with MS.[@B40] In-depth studies are needed to disclose the functional mechanism of these genes in MS.

It should be noted that since there is strong linkage disequilibrium (LD) at the HLA region, it is reasonable to infer that the significant signals for some of the genetic markers are partially due to their strong LD with true functional variants within the HLA region. Although the supplementary analysis could play a part in analyzing the function of causal variants, it is also necessary to research the functional mechanisms underlying the associations further, especially those between the HLAs and MS.

In conclusion, this gene-based GWAS has identified 20 novel MS-associated genes. The results highlight the advantages of gene-based association analysis over single SNP-based GWASs for detecting susceptibility genes for MS. The new findings may provide novel insights into the molecular mechanisms underlying MS.

The study was supported by Natural Science Foundation of China (81473046, 81401343, 31401079, 31271336, 31071097, and 81373010), the Natural Science Foundation of Jiangsu Province (BK20130300), the Startup Fund from Soochow University (Q413900112, Q413900712), the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry, and a Project of the Priority Academic Program Development of Jiangsu Higher Education Institutions.

**Conflicts of Interest:** The authors have no financial conflicts of interest.

Supplementary Materials
=======================

The online-only Data Supplement is available with this article at <http://dx.doi.org/10.3988/jcn.2015.11.4.311>.

###### Results of gene-based and differential expression analyses of 58 MS-associated genes

![](jcn-11-311-s001)

  Gene                               Chromosome   Start position   Length   \#SNP^\*^   Initial *p*    Genes significant in initial study based on SNP-based *p*   Genes in PheGenI   Replication *p*   Differential expression *p*   Sample^†^
  ---------------------------------- ------------ ---------------- -------- ----------- -------------- ----------------------------------------------------------- ------------------ ----------------- ----------------------------- -----------
  *C6orf10*                          6            32255475         89181    21          3.97×10^-39^   C6orf10                                                     C6orf10            3.16×10^-3^       NS                            NS
  *NOTCH4*                           6            32157620         39224    7           2.51×10^-31^   NOTCH4                                                      NOTCH4             NS                NS                            NS
  *TNXB*                             6            32003932         78221    2           6.61×10^-28^   TNXB                                                        TNXB               3.37×10^-3^       4.45×10^-2^                   S2
  *HLA-DRA*                          6            32402619         15204    9           3.73×10^-24^   HLA-DRA                                                     HLA-DRA            4.08×10^-3^       5.82×10^-5^                   S1
  *AIF1*                             6            31577994         11804    1           3.15×10^-23^   AIF1                                                        AIF1               NA                2.04×10^-2^                   S1
  *SNORA38*                          6            31585856         10131    1           3.15×10^-23^   SNORA38                                                     NA                 NA                NA                            NA
  *BAT2*                             6            31583450         27104    3           1.18×10^-22^   BAT2                                                        BAT2               3.42×10^-2^       NS                            NS
  *BAT3*                             6            31601805         23672    3           4.41×10^-19^   BAT3                                                        BAT3               9.52×10^-3^       NS                            NS
  *PPIAP9*                           6            31481654         11525    1           1.46×10^-15^   PPIAP9                                                      PPIAP9             4.67×10^-2^       NA                            NA
  *HLA-DQB2*                         6            32718875         17455    6           9.02×10^-15^   HLA-DQB2                                                    HLA-DQB2           1.01×10^-2^       NS                            NS
  *HLA-DQA2*                         6            32704163         15501    6           1.25×10^-14^   HLA-DQA2                                                    HLA-DQA2           3.93×10^-3^       NS                            NS
  *EHMT2*                            6            31842536         27928    1           8.91×10^-14^   EHMT2                                                       EHMT2              NS                4.30×10^-3^                   S1
                                                                                                                                                                                                        4.50×10^-2^                   S2
  *LOC100287272*                     6            31238352         13179    5           1.79×10^-13^   LOC100287272                                                NA                 4.76×10^-3^       NA                            NA
  *MCCD1*                            6            31491739         11269    2           4.39×10^-13^   MCCD1                                                       MCCD1              NA                NS                            NS
  *LOC100462812*                     6            31340494         10311    5           4.71×10^-13^   LOC100462812                                                NA                 8.07×10^-5^       NA                            NA
  *HCG27*                            6            31160537         16208    3           6.34×10^-13^   HCG27                                                       HCG27              3.84×10^-2^       NS                            NS
  *HLA-DRB9*                         6            32422597         10269    6           9.42×10^-13^   HLA-DRB9                                                    HLA-DRB9           1.60×10^-3^       NA                            NA
  *HLA-S*                            6            31344346         10918    6           4.89×10^-12^   HLA-S                                                       HLA-S              3.36×10^-2^       NA                            NA
  *HLA-DQA1*                         6            32600183         16246    1           1.68×10^-11^   HLA-DQA1                                                    HLA-DQA1           5.96×10^-4^       3.51×10^-2^                   S1
  *TAP2*                             6            32784610         26937    2           8.02×10^-11^   TAP2                                                        TAP2               NS                3.35×10^-3^                   S1
  *BTNL2*                            6            32357513         22387    7           1.43×10^-10^   BTNL2                                                       BTNL2              8.76×10^-5^       NS                            NS
  *HLA-DOB*                          6            32775540         14285    4           1.78×10^-10^   HLA-DOB                                                     HLA-DOB            NA                1.63×10^-5^                   S1
  *RDBP*                             6            31914864         17000    2           4.31×10^-10^   RDBP                                                        RDBP               4.16×10^-2^       2.01×10^-2^                   S1
                                                                                                                                                                                                        3.70×10^-2^                   S2
  *PSORS1C1*                         6            31077608         35261    10          8.59×10^-10^   PSORS1C1                                                    PSORS1C1           NS                4.85×10^-2^                   S2
  *HLA-DMB*                          6            32897406         16441    1           2.63×10^-9^    HLA-DMB                                                     HLA-DMB            NS                4.45×10^-2^                   S3
  *DHX16*                            6            30615896         29934    1           7.13×10^-9^    DHX16                                                       DHX16              2.73×10^-3^       NS                            NS
  *POU5F1*                           6            31127114         16337    4           9.96×10^-9^    POU5F1                                                      POU5F1             4.68×10^-3^       8.28×10^-3^                   S2
  *TCF19*                            6            31121303         15689    2           2.02×10^-7^    TCF19                                                       TCF19              1.78×10^-2^       NS                            NS
  *3.8-1.5 (HLA complex group 26)*   6            29727893         11180    4           2.40×10^-7^    3.8-1.5                                                     NA                 1.27×10^-3^       NA                            NA
  *HLA-F*                            6            29686117         13956    7           4.67×10^-7^    HLA-F                                                       NA                 1.17×10^-2^       2.64×10^-2^                   S1
  *LOC100507436*                     6            31362561         76025    8           4.98×10^-7^    LOC100507436                                                NA                 NS                NA                            NA
  *HLA-X*                            6            31424623         10644    1           1.55×10^-6^    NA                                                          HLA-X              NA                NA                            NA
  *HCG4P4*                           6            29917982         10428    1           8.60×10^-6^    NA                                                          HCG4P4             6.98×10^-4^       NA                            NA
  *RPL3P2*                           6            31243108         11240    2           1.66×10^-5^    NA                                                          RPL3P2             NA                NA                            NA
  *HLA-C*                            6            31231529         13326    2           1.72×10^-5^    NA                                                          HLA-C              4.29×10^-2^       6.15×10^-3^                   S1
                                                                                                                                                                                                        2.74×10^-2^                   S2
  *DHFRP2*                           6            31326244         13498    2           2.89×10^-5^    NA                                                          DHFRP2             NA                NA                            NA
  *MICE*                             6            29704234         17646    1           4.63×10^-5^    NA                                                          MICE               NA                NA                            NA
  *TRIM31*                           6            30065674         20193    1           1.18×10^-4^    NA                                                          TRIM31             1.31×10^-4^       NS                            NS
  *MOG*                              6            29619758         25391    2           1.21×10^-5^    NA                                                          NA                 5.09×10^-4^       2.12×10^-2^                   S1
                                                                                                                                                                                                        3.65×10^-2^                   S3
                                                                                                                                                                                                        2.92×10^-2^                   S4
  *HLA-DMA*                          6            32911391         14508    2           3.15×10^-7^    NA                                                          NA                 4.08×10^-2^       8.62×10^-4^                   S1
  *CCHCR1*                           6            31105216         25799    2           4.03×10^-5^    NA                                                          NA                 1.08×10^-3^       1.05×10^-2^                   S1
  *HCG22*                            6            31016984         15669    6           1.21×10^-4^    NA                                                          NA                 2.29×10^-2^       3.85×10^-2^                   S1
  *BRD2*                             6            32931437         22845    1           1.07×10^-5^    NA                                                          NA                 NS                5.65×10^-6^                   S1
  *FAM69A*                           1            93302721         129358   11          4.47×10^-5^    NA                                                          NA                 NA                1.63×10^-2^                   S1
  *LOC100294145*                     6            32856953         19582    3           2.27×10^-5^    NA                                                          NA                 NA                4.21×10^-2^                   S2
  *POU2F3*                           11           120000000        89702    3           1.13×10^-4^    NA                                                          NA                 NS                8.41×10^-3^                   S3
  *TRIM26P*                          6            30201078         13978    2           3.72×10^-5^    NA                                                          NA                 7.80×10^-6^       NA                            NA
  *HCP5P14*                          6            29733324         12339    1           1.33×10^-4^    NA                                                          NA                 9.94×10^-5^       NA                            NA
  *3.8-1.4(HLA complex group 26)*    6            29828692         11172    7           1.58×10^-5^    NA                                                          NA                 1.08×10^-3^       NA                            NA
  *C6orf15*                          6            31074000         11332    3           9.26×10^-5^    NA                                                          NA                 2.22×10^-3^       NS                            NS
  *MUC21*                            6            30946485         16190    4           4.40×10^-5^    NA                                                          NA                 3.42×10^-3^       NA                            NA
  *HLA-A*                            6            29905309         13352    4           1.61×10^-5^    NA                                                          NA                 8.38×10^-3^       NS                            NS
  *HLA-F-AS1*                        6            29689378         32448    7           1.83×10^-6^    NA                                                          NA                 2.52×10^-2^       NA                            NA
  *MXD3*                             5            177000000        16791    2           7.60×10^-7^    NA                                                          NA                 NS                NS                            NS
  *LOC100287247*                     22           \-               \-       1           2.71×10^-5^    NA                                                          NA                 NA                NA                            NA
  *LOC100127934*                     1            93392136         10509    1           6.18×10^-5^    NA                                                          NA                 NA                NA                            NA
  *RPL15P4*                          6            31490853         10645    1           2.08×10^-7^    NA                                                          NA                 NA                NA                            NA
  *HCG2P8*                           6            29767896         13543    1           3.96×10^-5^    NA                                                          NA                 NA                NA                            NA

PheGenI, Phenotype-Genotype Integrator.

^\*^The number of SNP included in a gene with *p*-value\<0.05, ^†^Ms-related cells sample used to differential expression ananlysis. S1: PBMC, S2: CD34+ HPC, S3: CD8+ T lymphocytes, S4, spinal cord.

The genes listed here that are not defined in the main text are chromosome 6 open reading frame 10 (*C6orf10*), notch 4 (*NOTCH4*), small nucleolar RNA, H/ACA box 38 (*SNORA38*), proline-rich coiled-coil 2A (*BAT2*), BCL2-associated athanogene 6 (*BAT3*), peptidylprolyl isomerase A pseudogene 9 (*PPIAP9*), major histocompatibility complex, class II, DQ beta 2 (*HLA-DQB2*), major histocompatibility complex, class II, DQ alpha 2 (*HLA-DQA2*), ubiquitin specific peptidase 8 pseudogene 1 (*LOC100287272*), mitochondrial coiled-coil domain 1 (*MCCD1*), fibroblast growth factor receptor 3 pseudogene 1 (*LOC100462812*), HLA complex group 27 (*HCG27*), major histocompatibility complex, class II, DR beta 9 (*HLA-DRB9*), major histocompatibility complex, class I, S (*HLA-S*), butyrophilin-like 2 (*BTNL2*), DEAH (*Asp-Glu-Ala-His*) box polypeptide 16 (*DHX16*) transcription factor 19 (*TCF19*), HLA complex group 26 pseudogene (3.8-1.5), MHC class I polypeptide-related sequence A (*LOC100507436*), major histocompatibility complex, class I, x (*HLA-X*), HLA complex group 4 pseudogene 4 (*HCG4P4*), ribosomal protein L3 pseudogene 2 (*RPL3P2*), dihydrofolate reductase pseudogene 2 (*DHFRP2*), MHC class I polypeptide-related sequence E (*MICE*), ripartite motif containing 31 (*TRIM31*), MAX dimerization protein 3 (*MXD3*), similar to hCG1987428 (*LOC100287247*), meiotic nuclear divisions 1 homolog (*LOC100127934*), ribosomal protein L15 pseudogene 4 (*RPL15P4*), HLA complex group 2 pseudogene 8 (*HCG2P8*) respectively.

###### Enrichment of GO terms and KEGG pathways of the 58 MS-associated genes

![](jcn-11-311-s002)

  Category          Term                                                                                                    Count   \%      Genes                                                                                                                                                                                                   List total   Relative enrichment   Bonferroni^\*^
  ----------------- ------------------------------------------------------------------------------------------------------- ------- ------- ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ------------ --------------------- ----------------
  GOTERM_BP_FAT     GO:0019882\~antigen processing and presentation                                                         12      21.43   3112, 285830, 3117, 3118, 6891, 3107, 3109, 3108, 3134, 3105, 3132, 3122                                                                                                                                25           78.23                 1.09×10^-16^
  GOTERM_CC_FAT     GO:0042611\~MHC protein complex                                                                         11      19.64   3112, 285830, 3117, 3118, 3107, 3109, 3108, 3134, 3105, 3132, 3122                                                                                                                                      24           102.78                1.06×10^-16^
  SP_PIR_KEYWORDS   mhc ii                                                                                                  9       16.07   3112, 3117, 3118, 3107, 3109, 3108, 3105, 3132, 3122                                                                                                                                                    34           164.25                1.84×10^-14^
  UP_SEQ_FEATURE    Region of interest:Connecting peptide                                                                   9       16.07   3112, 3117, 3118, 3107, 3109, 3108, 3134, 3105, 3122                                                                                                                                                    33           137.17                3.39×10^-13^
  UP_SEQ_FEATURE    Domain:Ig-like C1-type                                                                                  9       16.07   3112, 3117, 3118, 3107, 3109, 3108, 3134, 3105, 3122                                                                                                                                                    33           130.32                5.42×10^-13^
  KEGG_PATHWAY      hsa05330:Allograft rejection                                                                            9       16.07   3112, 3117, 3118, 3107, 3109, 3108, 3134, 3105, 3122                                                                                                                                                    14           90.80                 7.23×10^-14^
  KEGG_PATHWAY      hsa05332:Graft-versus-host disease                                                                      9       16.07   3112, 3117, 3118, 3107, 3109, 3108, 3134, 3105, 3122                                                                                                                                                    14           83.82                 1.47×10^-13^
  KEGG_PATHWAY      hsa04940:Type I diabetes mellitus                                                                       9       16.07   3112, 3117, 3118, 3107, 3109, 3108, 3134, 3105, 3122                                                                                                                                                    14           77.83                 2.80×10^-13^
  KEGG_PATHWAY      hsa04612:Antigen processing and presentation                                                            10      17.86   3112, 3117, 3118, 6891, 3107, 3109, 3108, 3134, 3105, 3122                                                                                                                                              14           43.76                 7.53×10^-13^
  KEGG_PATHWAY      hsa05320:Autoimmune thyroid disease                                                                     9       16.07   3112, 3117, 3118, 3107, 3109, 3108, 3134, 3105, 3122                                                                                                                                                    14           64.10                 1.50×10^-12^
  INTERPRO          IPR003597:Immunoglobulin C1-set                                                                         9       16.07   3112, 3117, 3118, 3107, 3109, 3108, 3134, 3105, 3122                                                                                                                                                    29           76.03                 1.16×10^-11^
  UP_SEQ_FEATURE    Region of interest:Alpha-1                                                                              7       12.50   3117, 3118, 3107, 3108, 3134, 3105, 3122                                                                                                                                                                33           202.71                1.56×10^-10^
  UP_SEQ_FEATURE    Region of interest: Alpha-2                                                                             7       12.50   3117, 3118, 3107, 3108, 3134, 3105, 3122                                                                                                                                                                33           202.71                1.56×10^-10^
  INTERPRO          IPR014745:MHC class II, alpha/beta chain, N-terminal                                                    7       12.50   3112, 3117, 3118, 3109, 3108, 3132, 3122                                                                                                                                                                29           182.78                7.35×10^-11^
  INTERPRO          IPR003006:Immunoglobulin/major histocompatibility complex, conserved site                               9       16.07   3112, 3117, 3118, 3107, 3109, 3108, 3134, 3105, 3122                                                                                                                                                    29           59.43                 8.98×10^-11^
  KEGG_PATHWAY      hsa05416:Viral myocarditis                                                                              9       16.07   3112, 3117, 3118, 3107, 3109, 3108, 3134, 3105, 3122                                                                                                                                                    14           46.04                 2.47×10^-11^
  SMART             SM00407:IGc1                                                                                            9       16.07   3112, 3117, 3118, 3107, 3109, 3108, 3134, 3105, 3122                                                                                                                                                    24           50.07                 7.27×10^-11^
  GOTERM_CC_FAT     GO:0042613\~MHC class II protein complex                                                                7       12.50   3112, 3117, 3118, 3109, 3108, 3132, 3122                                                                                                                                                                24           128.55                5.56×10^-10^
  GOTERM_BP_FAT     GO:0002504\~antigen processing and presentation of peptide or polysaccharide antigen via MHC class II   7       12.50   3112, 3117, 3118, 3109, 3108, 3132, 3122                                                                                                                                                                25           114.78                3.42×10^-9^
  GOTERM_MF_FAT     GO:0032395\~MHC class II receptor activity                                                              6       10.71   3112, 3117, 3118, 3107, 3108, 3122                                                                                                                                                                      23           178.26                7.85×10^-9^
  SP_PIR_KEYWORDS   Immune response                                                                                         10      17.86   3112, 3117, 3118, 6891, 3107, 3109, 3108, 3134, 3105, 3122                                                                                                                                              34           25.26                 8.42×10^-9^
  KEGG_PATHWAY      hsa04514:Cell adhesion molecules (CAMs)                                                                 9       16.07   3112, 3117, 3118, 3107, 3109, 3108, 3134, 3105, 3122                                                                                                                                                    14           24.76                 4.04×10^-9^
  GOTERM_CC_FAT     GO:0042825\~TAP complex                                                                                 5       8.93    3112, 6891, 3109, 3108, 3122                                                                                                                                                                            24           380.42                2.00×10^-8^
  GOTERM_CC_FAT     GO:0042824\~MHC class I peptide loading complex                                                         5       8.93    3112, 6891, 3109, 3108, 3122                                                                                                                                                                            24           295.88                7.18×10^-8^
  GOTERM_BP_FAT     GO:0048002\~antigen processing and presentation of peptide antigen                                      6       10.71   6891, 3107, 3108, 3134, 3105, 3122                                                                                                                                                                      25           115.95                2.17×10^-7^
  PIR_SUPERFAMILY   PIRSF001991:Class II histocompatibility antigen                                                         6       10.71   3112, 3117, 3118, 3109, 3108, 3122                                                                                                                                                                      18           94.82                 2.79×10^-8^
  INTERPRO          IPR007110:Immunoglobulin-like                                                                           11      19.64   3112, 3117, 3118, 4340, 3107, 3109, 3108, 3134, 56244, 3105, 3122                                                                                                                                       29           12.61                 3.53×10^-7^
  KEGG_PATHWAY      hsa05310:Asthma                                                                                         6       10.71   3112, 3117, 3118, 3109, 3108, 3122                                                                                                                                                                      14           75.15                 1.10×10^-7^
  GOTERM_BP_FAT     GO:0006955\~immune response                                                                             12      21.43   3112, 285830, 3117, 3118, 6891, 3107, 3109, 3108, 3134, 3105, 3132, 3122                                                                                                                                25           9.41                  1.53×10^-6^
  GOTERM_MF_FAT     GO:0042288\~MHC class I protein binding                                                                 5       8.93    3112, 6891, 3109, 3108, 3122                                                                                                                                                                            23           176.40                8.88×10^-7^
  INTERPRO          IPR013783:Immunoglobulin-like fold                                                                      11      19.64   3112, 3117, 3118, 4340, 3107, 3109, 3108, 3134, 56244, 3105, 3122                                                                                                                                       29           11.43                 9.06×10^-7^
  GOTERM_MF_FAT     GO:0042287\~MHC protein binding                                                                         5       8.93    3112, 6891, 3109, 3108, 3122                                                                                                                                                                            23           112.90                6.11×10^-6^
  KEGG_PATHWAY      hsa04672:Intestinal immune network for IgA production                                                   6       10.71   3112, 3117, 3118, 3109, 3108, 3122                                                                                                                                                                      14           44.48                 1.72×10^-6^
  SP_PIR_KEYWORDS   Heterodimer                                                                                             6       10.71   3117, 3118, 3107, 3134, 3105, 3122                                                                                                                                                                      34           32.96                 6.98×10^-5^
  SP_PIR_KEYWORDS   Transmembrane protein                                                                                   10      17.86   3112, 3117, 3118, 4340, 6891, 3107, 3108, 3134, 3105, 3122                                                                                                                                              34           8.81                  7.59×10^-5^
  INTERPRO          IPR001003:MHC class II, alpha chain, N-terminal                                                         4       7.14    3117, 3118, 3108, 3122                                                                                                                                                                                  29           191.48                7.32×10^-5^
  KEGG_PATHWAY      hsa05322:Systemic lupus erythematosus                                                                   6       10.71   3112, 3117, 3118, 3109, 3108, 3122                                                                                                                                                                      14           22.01                 6.04×10^-5^
  GOTERM_BP_FAT     GO:0002474\~antigen processing and presentation of peptide antigen via MHC class I                      4       7.14    6891, 3107, 3134, 3105                                                                                                                                                                                  25           127.32                6.60×10^-4^
  GOTERM_CC_FAT     GO:0044459\~plasma membrane part                                                                        15      26.79   4855, 3112, 3117, 3118, 6891, 394263, 285830, 199, 3107, 3109, 3108, 3134, 3105, 3132, 3122                                                                                                             24           3.63                  2.43×10^-4^
  INTERPRO          IPR001039:MHC class I, alpha chain, alpha1, and alpha2                                                  4       7.14    285830, 3107, 3134, 3105                                                                                                                                                                                29           85.10                 9.56×10^-4^
  INTERPRO          IPR011161:MHC class I-like antigen recognition                                                          4       7.14    285830, 3107, 3134, 3105                                                                                                                                                                                29           82.06                 1.07×10^-3^
  GOTERM_CC_FAT     GO:0042612\~MHC class I protein complex                                                                 4       7.14    285830, 3107, 3134, 3105                                                                                                                                                                                24           76.08                 1.17×10^-3^
  BIOCARTA          h_mhcPathway:Antigen processing and presentation                                                        3       5.36    6891, 3105, 3122                                                                                                                                                                                        3            143.70                5.67×10^-4^
  UP_SEQ_FEATURE    Sequence variant                                                                                        31      55.36   170679, 3112, 4855, 11074, 54535, 6891, 56244, 7148, 10919, 6941, 199, 6046, 83463, 5460, 3107, 3109, 3108, 3134, 3105, 401250, 3117, 3118, 4340, 29113, 8449, 10665, 7916, 394263, 25833, 3122, 7917   33           1.50                  1.94×10^-2^
  SP_PIR_KEYWORDS   Polymorphism                                                                                            31      55.36   170679, 3112, 4855, 11074, 54535, 6891, 56244, 7148, 10919, 6941, 199, 6046, 83463, 5460, 3107, 3109, 3108, 3134, 3105, 401250, 3117, 3118, 4340, 29113, 8449, 10665, 7916, 394263, 25833, 3122, 7917   34           1.52                  7.47×10^-3^
  UP_SEQ_FEATURE    Region of interest:Alpha-3                                                                              3       5.36    3107, 3134, 3105                                                                                                                                                                                        33           193.06                2.92×10^-2^
  SP_PIR_KEYWORDS   mhc i                                                                                                   3       5.36    3107, 3134, 3105                                                                                                                                                                                        34           169.72                1.05×10^-2^
  GOTERM_BP_FAT     GO:0002478\~antigen processing and presentation of exogenous peptide antigen                            3       5.36    6891, 3108, 3122                                                                                                                                                                                        25           147.58                3.25×10^-2^
  PIR_SUPERFAMILY   PIRSF001990:Class I histocompatibility antigen                                                          3       5.36    3107, 3134, 3105                                                                                                                                                                                        18           136.96                2.30×10^-3^
  GOTERM_BP_FAT     GO:0019884\~antigen processing and presentation of exogenous antigen                                    3       5.36    6891, 3108, 3122                                                                                                                                                                                        25           115.95                5.30×10^-2^
  GOTERM_MF_FAT     GO:0042277\~peptide binding                                                                             5       8.93    3112, 6891, 3109, 3108, 3122                                                                                                                                                                            23           13.90                 2.69×10^-2^
  INTERPRO          IPR000353:MHC class II, beta chain, N-terminal                                                          3       5.36    3112, 3109, 3132                                                                                                                                                                                        29           101.37                2.84×10^-2^
  GOTERM_MF_FAT     GO:0032393\~MHC class I receptor activity                                                               3       5.36    3107, 3134, 3105                                                                                                                                                                                        23           99.61                 2.89×10^-2^
  GOTERM_CC_FAT     GO:0005886\~plasma membrane                                                                             16      28.57   4855, 3112, 3117, 3118, 4340, 6891, 394263, 285830, 199, 3107, 3109, 3108, 3134, 3105, 3132, 3122                                                                                                       24           2.26                  2.98×10^-2^

Count, the number of genes enriched in particular GO terms or KEGG pathways; Genes, potential genes enriched in particular GO terms or KEGG pathways.

^\*^*p* value with Bonferroni correction for multiple tests. Only significant results with *p*\<0.05 are listed.

GO: Gene Ontology, KEGG: Kyoto Encyclopedia of Genes and Genomes, MHC: major histocompatibility complex, MS: mltiple sclerosis.

![Quantile-quantile plots of the association results for the initial genome scan. The tail of the distribution of gene-based probability values deviated more significantly than those of SNPs inside or outside of the gene, which suggests that the power was higher for a gene-based association analysis than a single SNP-based GWAS in detecting associations. GWAS: genome-wide association study, SNP: single-nucleotide polymorphism.](jcn-11-311-g001){#F1}

![Manhattan plot of gene probability values on chromosomes. Most of the genes significantly associated with MS are mapped to the HLA region (chromosome 6). HLA: human leukocyte antigen, MS: multiple sclerosis.](jcn-11-311-g002){#F2}

![Protein-protein interactions between MS-associated genes. Only the connected genes are shown. The novel genes are labeled in red. Different line colors represent the types of evidence for the association. HLA class I cluster: major histocompatibility complex, class I, A (*HLA-A*), Major histocompatibility complex, class I, C (*HLA-C*), and major histocompatibility complex, class I, F (*HLA-F*); HLA class II cluster: major histocompatibility complex, class II, DQ alpha 1 (*HLA-DQA1*), major histocompatibility complex, class II, DQ alpha 2 (*HLA-DQA2*), major histocompatibility complex, class II, DQ beta 2 (*HLA-DQB2*), major histocompatibility complex, class II, DR alha (*HLA-DRA*), major histocompatibility complex, class II, DO beta (*HLA-DOB*), major histocompatibility complex, class II, DM alpha (*HLA-DMA*), and major histocompatibility complex, class II, DM beta (*HLA-DMB*). HLA: human leukocyte antigen, MS: Multiple sclerosis.](jcn-11-311-g003){#F3}

###### MS-associated genes with significantly differential expressions in MS-related cells/tissue

![](jcn-11-311-i001)

  Sample                       S1                                    S2                           S3                                    S4
  ---------------------------- ------------------------------------- ---------------------------- ------------------------------------- -------------------------------------
  Target cells/tissue          PBMC                                  CD34+ HPC                    CD8+ T lymphocytes                    Spinal cord
  Sample size                  12:15                                 8:5                          4:4                                   8:8
  Platform                     \[HG-U133_Plus_2\] Affymetrix Human   Agilent-014850 Whole Human   \[HG-U133_Plus_2\] Affymetrix Human   \[HG-U133_Plus_2\] Affymetrix Human
  Genome U133 Plus 2.0 Array   Genome Microarray 4×44K G4112F        Genome U133 Plus 2.0 Array   Genome U133 Plus 2.0 Array            
  PMID                         22021740                              22252466                     21216829                              24910450
  GSE No.                      GSE21942                              GSE27694                     GSE16461                              GSE52139

     Gene         Probe ID^\*^   *t*-test, *p*   Gene             Probe ID^\*^   *t*-test, *p*   Gene        Probe ID^\*^   *t*-test, *p*   Gene    Probe ID^\*^    *t*-test, *p*
  -- ------------ -------------- --------------- ---------------- -------------- --------------- ----------- -------------- --------------- ------- --------------- ---------------
     *EHMT2*      207484_s\_at   4.30×10^-31^    *EHMT2*          A_24_P801451   4.50×10^-2^     \-          \-             \-              \-      \-              \-
     *HLA-C*      211799_x\_at   6.15×10^-3^     *HLA-C*          A_24_P298409   2.74×10^-2^     \-          \-             \-              \-      \-              \-
     *RDBP*       209219_at      2.01×10^-2^     *RDBP*           A_23_P122545   3.70×10^-2^     \-          \-             \-              \-      \-              \-
     *MOG*        214650_x\_at   2.12×10^-2^     \-               \-             \-              *MOG*       214650_x\_at   3.65×10^-2^     *MOG*   1555807_a\_at   2.92×10^-2^
     *BRD2*       208685_x\_at   5.65×10^-6^     *POU5F1*         A_24_P144601   8.28×10^-3^     *POU2F3*    215355_at                                              
     *HLA-DOB*    205671_s\_at   1.63×10^-5^     *LOC100294145*   A_32_P97739    4.21×10^-2^     *HLA-DMB*   203932_at                                              
     *HLA-DRA*    208894_at      5.82×10^-5^     *TNXB*           A_24_P911362   4.45×10^-2^                                                                        
     *HLA-DMA*    217478_s\_at   8.62×10^-4^     *PSORS1C1*       A_24_P24848    4.85×10^-2^                                                                        
     *TAP2*       204770_at      3.35×10^-3^                                                                                                                        
     *AIF1*       209901_x\_at   2.04×10^-2^                                                                                                                        
     *HLA-F*      204806_x\_at   2.64×10^-2^                                                                                                                        
     *HLA-DQA1*   236203_at      3.51×10^-2^                                                                                                                        
     *HCG22*      1560767_at     3.85×10^-2^                                                                                                                        
     *CCHCR1*     37424_at       1.05×10^-2^                                                                                                                        
     *FAM69A*     1556498_at     1.63×10^-2^                                                                                                                        

The ratio listed in the line of \"Sample size\" is the number of multiple sclerosis (MS) cases compared with that of controls. The genes listed here that are not defined in the main text are major histocompatibility complex, class II, DO beta (*HLA-DOB*), major histocompatibility complex, class II, DR alha (*HLA-DRA*), POU class 5 homeobox 1 (*POU5F1*), uncharacterized LOC100294145 (*LOC100294145*), tenascin XB (*TNXB*), psoriasis susceptibility 1 candidate 1 (*PSORS1C1*), major histocompatibility complex, class II, DM beta (*HLA-DMB*), transporter 2, ATP-binding cassette, sub-family B (*TAP2*), allograft inflammatory factor 1 (*AIF1*), major histocompatibility complex, class I, F (*HLA-F*), major histocompatibility complex, class II, DQ alpha 1 (*HLA-DQA1*), respectively.

^\*^Only the most significant probe is listed, even if more than one probe was detected for a gene.

GSE No.: Gene Expression Omnibus number, [www.ncbi.nlm.nih.gov/geo/](www.ncbi.nlm.nih.gov/geo/), PMID: PubMed unique identifier.

###### Fifteen novel MS-associated genes, supported by replication studies and/or expression studies

![](jcn-11-311-i002)

  Gene                               Chromosome   Start position   Length   \#SNP^\*^   Initial *p*   Replication *p*   Differential expression *p*, sample^†^   
  ---------------------------------- ------------ ---------------- -------- ----------- ------------- ----------------- ---------------------------------------- ----
  *MOG*                              6            29619758         25391    2           1.21×10^-5^   5.09×10^-4^       2.12×10^-2^                              S1
  3.65×10^-2^                        S3                                                                                                                          
  2.92×10^-2^                        S4                                                                                                                          
  *HLA-DMA*                          6            32911391         14508    2           3.15×10^-7^   4.08×10^-2^       8.62×10^-4^                              S1
  *CCHCR1*                           6            31105216         25799    2           4.03×10^-5^   1.08×10^-3^       1.05×10^-2^                              S1
  *HCG22*                            6            31016984         15669    6           1.21×10^-4^   2.29×10^-2^       3.85×10^-2^                              S1
  *BRD2*                             6            32931437         22845    1           1.07×10^-5^   NS                5.65×10^-6^                              S1
  *FAM69A*                           1            93302721         129358   11          4.47×10^-5^   NA                1.63×10^-2^                              S1
  *LOC100294145*                     6            32856953         19582    3           2.27×10^-5^   NA                4.21×10^-2^                              S2
  *POU2F3*                           11           120000000        89702    3           1.13×10^-4^   NS                8.41×10^-3^                              S3
  *TRIM26P*                          6            30201078         13978    2           3.72×10^-5^   7.80×10^-6^       NA                                       NA
  *HCP5P14*                          6            29733324         12339    1           1.33×10^-4^   9.94×10^-5^       NA                                       NA
  *3.8-1.4 (HLA complex group 26)*   6            29828692         11172    7           1.58×10^-5^   1.08×10^-3^       NA                                       NA
  *C6orf15*                          6            31074000         11332    3           9.26×10^-5^   2.22×10^-3^       NS                                       NS
  *MUC21*                            6            30946485         16190    4           4.40×10^-5^   3.42×10^-3^       NA                                       NA
  *HLA-A*                            6            29905309         13352    4           1.61×10^-5^   8.38×10^-3^       NS                                       NS
  *HLA-F-AS1*                        6            29689378         32448    7           1.83×10^-6^   2.52×10^-2^       NA                                       NA

The genes listed here that are not defined in the main text are tripartite motif containing 26 B (*TRIM26P*), HLA complex P5 pseudogene 14 (*HCP5P14*), HLA complex group 26 pseudogene (3.8-1.4), mucin 21, cell surface associated (*MUC21*), HLA-F antisense RNA 1 (*HLA-F-AS1*), respectively.

^\*^Number of SNPs included in the gene with *p*\<0.05, ^†^Multiple sclerosis (MS)-related cells/tissue sample used for the differential expression analysis; S1, PBMC; S2, CD34+ HPC; S3, CD8+ T lymphocytes; S4, spinal cord.

NA: not available, NS: not significant.
