Genetic Determinants of Antibody Levels in Cerebrospinal Fluid in Multiple Sclerosis: Possible Links to Endogenous Retroviruses

International Journal of Molecular Sciences

Brief Report Genetic Determinants of Antibody Levels in Cerebrospinal Fluid in Multiple Sclerosis: Possible Links to Endogenous Retroviruses

Alexander Emmer 1,*, Christine Brütting 1,2, Malte Kornhuber 1 and Martin S. Staege 2

1 Department of Neurology, Martin Luther University Halle-Wittenberg, Ernst-Grube-Str. 40, 06120 Halle (Saale), Germany; [email protected] (C.B.); [email protected] (M.K.) 2 Department of Surgical and Conservative Pediatrics and Adolescent Medicine, Martin Luther University Halle-Wittenberg, Ernst-Grube-Str. 40, 06120 Halle (Saale), Germany; [email protected] * Correspondence: [email protected]; Tel.: +49-345-557-2738; Fax: +49-345-557-3335

Received: 14 February 2018; Accepted: 7 March 2018; Published: 9 March 2018

Abstract: The pathogenesis of multiple sclerosis (MS) has not been clarified. In addition to environmental factors; genetic determinants have been implicated in the pathogenesis of MS. Furthermore, endogenous retroviruses (ERV) might play a role in MS. The presence of oligoclonal immunoglobulin in cerebrospinal fluid (CSF) is a typical feature of MS. Recently, genetic polymorphisms in loci on human chromosomes 6, 14 and 18 have been identified as major determinants of CSF antibody levels in MS. The functional relevance of these single nucleotide polymorphisms (SNPs) remains unclear and none of them is located in an open reading frame. In previous studies, we identified ERV sequences in the vicinity of MS associated SNPs. Here, we describe the identification of ERV sequences in the neighborhood of SNPs associated with CSF antibody levels. All of the identified SNPs are located in the vicinity of ERV sequences. One of these sequences has very high homology to a sequence derived from the so-called MS-associated retrovirus (MSRV). Another cluster of three ERV sequences from the immunoglobulin heavy chain locus has retained the typical organization of retroviral genomes. These observations might shed new light on a possible association between ERVs and MS pathogenesis.

Keywords: multiple sclerosis; oligoclonal bands; immunoglobulin G index; genetic determinants; endogenous retrovirus (ERV)

1. Introduction Multiple sclerosis (MS) is a disease that is characterized by central nervous system demyelination with concomitant destruction of neurons. The causes of MS are unknown. However, during the course of MS, an activation of the immune system takes place and inflammation plays a major role in disease activity. The presence of oligoclonal immunoglobulin in the cerebrospinal fluid (CSF) is a typical feature of MS and so-called oligoclonal bands (OCBs), as detected by isoelectric focusing, are of diagnostic importance in MS [1]. The pathogenesis of MS OCB is still unresolved. OCBs have also been reported in the CSF of patients with different viral diseases like e.g., in human immunodeficiency virus (HIV) infected individuals [2–4]. Recently, genetic polymorphisms in loci on human chromosomes 6, 14 and 18 have been identified as major determinants of CSF antibody levels in MS [5]. The functional relevance of these single nucleotide polymorphisms (SNPs) remains unclear and none of them is located in an open reading frame. Previously, we have put forward the hypothesis that inflammation in MS, including humoral parts, could be driven by one or more endogenous retrovirus (ERV) elements [6]. If so, MS OCB should be somehow linked to ERV loci in the human genome. If ERV elements do have an impact on the manifestation of OCB in MS, their genomic representation might be related to

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Int. J. Mol. Sci. 2018, 19, 786 2 of 8 the human genome. If ERV elements do have an impact on the manifestation of OCB in MS, their genomic representation might be related to identified SNP loci [7]. Therefore, we were interested in identifiedwhether the SNP identified loci [7]. Therefore,genetic determinants we were interested with high in whetherand reproducible the identified impact genetic on the determinants presence of withautochthonous high and reproducible IgG synthesis impact in MS onpatients the presence would fit of this autochthonous hypothesis. IgG synthesis in MS patients would fit this hypothesis. 2. Results 2. Results On chromosome 18 (nucleotide accession number NC_000018.10:49997791-51997791), we found 16 largeOn chromosomeopen reading 18 frames (nucleotide (ORFs) accession in the two number megabase NC_000018.10:49997791-51997791), pairs surrounding the SNP rs9807334. we found Only 16 largeone of open these reading ORFs frames showed (ORFs) high in sequence the two megabase similarity pairs to surroundingproteins from the re SNPtro-transcribing rs9807334. Only viruses one of(Figure these ORFs1, Supplementary showed high Table sequence S1). similarityInterestingly, to proteins this ORF from encodes retro-transcribing a polymerase viruses with very (Figure high1, Supplementaryhomology (expect Table value S1). 0.0) Interestingly, to a sequence this ORF identi encodesfied ina the polymerase so-called withMS-associated very high homologyretrovirus (expect(MSRV) value [8]. A 0.0)similar to asequence sequence of identical identified length in the is so-calledpresent on MS-associated chromosome 12 retrovirus in the human (MSRV) genome [8]. A(Figure similar 2). sequence of identical length is present on chromosome 12 in the human genome (Figure2).

Figure 1. Schematic organization of the locus on human chromosome 18 including rs9807334. The two Figure 1. Schematic organization of the locus on human chromosome 18 including rs9807334. megabase pairs flanking rs9807334 were analyzed using getorf for the presence of open reading The two megabase pairs flanking rs9807334 were analyzed using getorf for the presence of open frames (ORFs) with a minimal length of one kilobase. Sixteen ORFs were identified (see reading frames (ORFs) with a minimal length of one kilobase. Sixteen ORFs were identified (seeSupplementarySupplementary Table Table S1). S1These). These ORFs ORFs were were analyz analyzeded using using BLASTP BLASTP against against the database the database of retro- of retro-transcribingtranscribing viruses viruses (taxid (taxid:35268).:35268). One ORF One ORFwith withhigh highsequence sequence similarity similarity to retroviruses to retroviruses was wasidentified. identified. This ThisORF ORFis located is located between between the SMAD the SMAD family family member member 4 (SMAD4) 4 (SMAD4) locus and locus the and Mex-3 the Mex-3homolog homolog C (MEX3C) C (MEX3C) locus locus(for (fora co amplete complete list listof ofall allgenes genes in in this this chromosomal chromosomal region seesee SupplementarySupplementary Table Table S2. S2. ELAC1:ELAC1: elaC-likeelaC-like shortshort formform ofof RNaseRNase Z;Z; MAPK4:MAPK4: mitogen-activated proteinprotein kinasekinase 4;4; MEX3C: MEX3C: mex-3 mex-3 RNA RNA binding binding family family member member C; SMAD4: C; SMAD4: small bodysmall sizebody and size mothers and mothers against decapentaplegicagainst decapentaplegic homolog homolog 4. Arrows 4. indicateArrows indicate the direction the direction of the ORFs. of the ORFs.

OnOn chromosomechromosome 1414 (NC_000014.9:104706543-106706543),(NC_000014.9:104706543-106706543), wewe foundfound aa largelarge numbernumber ofof ORFsORFs surroundingsurrounding rs11621145rs11621145 (Supplementary (Supplementary Table Table S3). S3). From From these these ORFs, ORFs, nine nine showed showed some some similarity similarity to × × × retroviralto retroviral proteins proteins (2 (2polymerase × polymerase (pol), (pol), 3 group3 × group specific specific antigen antigen (gag), (gag), 3 envelope 3 × envelope protein protein (env), × and(env), 1 andHIV 1 negative× HIV negative regulatory regulatory factor (nef)). factor Most (nef)). of these Most ORFs of these showed ORFs only showed low sequence only low similarities sequence tosimilarities retro-transcribing to retro-transcribing viruses. However, viruses. a clusterHowever, of three a cluster large ORFsof three (ORF85-87 large ORFs in Supplementary (ORF85-87 in TableSupplementary S3) encodes Table putative S3) encodes polypeptides putative with polypeptid high homologieses with high to gag,homologies pol, and to env gag, proteins pol, and from env retroviruses,proteins from respectively. retroviruses, These respectively. three ORFs These showed three the typicalORFs showed topology the of thetypical gag-pol-env topology organization of the gag-

Int. J. Mol. Sci. 2018, 19, 786 3 of 8 Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 3 of 7 ofpol-env retroviruses organization and are located of retroviruses between the and immunoglobulin are located between heavy chain the variable immunoglobulin region genes heavy IGHV3-19 chain andvariable IGHV3-20 region (Figure genes3 ).IGHV3-19 and IGHV3-20 (Figure 3).

FigureFigure 2. 2.Multiple Multiple sequence sequence alignment alignment of of MS MS associated associated retrovirus retrovirus MSRV MSRV and and predicted predicted ORFs ORFs from from chromosomeschromosomes 12 12 and and 18. The18. twoThe megabasetwo megabase pairs flankingpairs flanking rs9807334 rs9807334 on chromosome on chromosome 18 were analyzed 18 were usinganalyzed getorf using for the getorf presence for the of presence open reading of open frames reading (ORFs) frames with (ORFs) a minimal with lengtha minimal of one length kilobase. of one Sixteenkilobase. ORFs Sixteen were ORFs identified were (see identified Supplementary (see Supplemen Table S1).tary These Table ORFsS1). These were OR analyzedFs were using analyzed BLASTP using againstBLASTP the against database the of database retro-transcribing of retro-transcribing viruses (taxid:35268). viruses (taxid:35268). One ORF with One high ORF sequence with high similarity sequence tosimilarity retroviruses to retroviruses was identified. was Presentedidentified. is Presen an alignmentted is an of alignment this ORF (ORF14)of this ORF and a(ORF14) polyprotein and a sequencepolyprotein from sequence the multiple from sclerosis the multiple associated sclerosis retrovirus associated (MSRV; accessionretrovirus number(MSRV; AAB66528.1:6-379) accession number andAAB66528.1:6-379) a similar sequence and from a chromosomesimilar sequence 12 (Chr.12; from accession chromosome number 12 EAW96383.1). (Chr.12; accession number EAW96383.1). In the major histocompatibility complex region on chromosome 6, a combination of SNPs has In the major histocompatibility complex region on chromosome 6, a combination of SNPs has been associated with CSF immunoglobulin levels [5]. Therefore, we included the complete region been associated with CSF immunoglobulin levels [5]. Therefore, we included the complete region one one megabase pairs upstream and one megabase pairs downstream of the rs6457617 and rs9271640 megabase pairs upstream and one megabase pairs downstream of the rs6457617 and rs9271640 in our in our analysis (accession number NC_000006.12:31624423-33696074). We found 27 open reading analysis (accession number NC_000006.12:31624423-33696074). We found 27 open reading frames frames surrounding the two polymorphisms (Supplementary Table S5). Only one of these ORFs surrounding the two polymorphisms (Supplementary Table S5). Only one of these ORFs showed showed homology to retroviral sequences. This ORF (ORF15 in Supplementary Table S5) showed high homology to retroviral sequences. This ORF (ORF15 in Supplementary Table S5) showed high homology to envelope proteins from endogenous retroviruses. homology to envelope proteins from endogenous retroviruses.

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Figure 3. Schematic organization of the immunoglobulin locus including rs11621145 on human Figure 3. Schematic organization of the immunoglobulin locus including rs11621145 on human chromosome 14. The two megabase pairs flanking rs11621145 were analyzed using getorf for the chromosome 14. The two megabase pairs flanking rs11621145 were analyzed using getorf for the presence of open reading frames (ORFs) with a minimal length of one kilobase. One hundred and presence of open reading frames (ORFs) with a minimal length of one kilobase. One hundred and sixty-nine ORFs were identified (see Supplementary Table S3). These ORFs were analyzed using sixty-nine ORFs were identified (see Supplementary Table S3). These ORFs were analyzed using BLASTPBLASTP againstagainst thethe databasedatabase ofof retro-transcribingretro-transcribing virusesviruses (taxid:35268).(taxid:35268). NineNine ORFsORFs withwith sequencesequence similaritysimilarity toto retrovirusesretroviruses werewere identified.identified. AA clustercluster ofof threethree ORFsORFs formingforming aa retrovirus-likeretrovirus-like compositecomposite elementelement isis locatedlocated betweenbetween thethe HOMERHOMER homologhomolog 22 pseudogenepseudogene 22 andand thethe solutesolute carriercarrier familyfamily 2020 membermember 11 pseudogenepseudogene 22 inin thethe immunoglobulinimmunoglobulin heavyheavy chainchain (IGH)(IGH) locus.locus. For aa completecomplete listlist ofof allall genesgenes in this chromosomal chromosomal region, region, see see Supplementary Supplementary Table Table S4. S4. Arrows Arrows indicate indicate the the direction direction of the of theORFs. ORFs.

3. Discussion 3. Discussion Genome wide association studies currently enlarge our knowledge and understanding of Genome wide association studies currently enlarge our knowledge and understanding of disease pathogenesis [9]. However, the exact functions and especially the interactions between disease pathogenesis [9]. However, the exact functions and especially the interactions between disease-associated polymorphisms have not been completely elucidated. Genetic determinants of disease-associated polymorphisms have not been completely elucidated. Genetic determinants of antibody levels in the CSF of patients with MS have been identified in a couple of loci that were antibody levels in the CSF of patients with MS have been identified in a couple of loci that were associated with the presence of OCB or elevated IgG indices [5]. associated with the presence of OCB or elevated IgG indices [5]. Polymorphisms on chromosomes 6, 14 and 18 showed the strongest association with OCB and/or Polymorphisms on chromosomes 6, 14 and 18 showed the strongest association with OCB and/or immunoglobulin indices [5]. The signal from chromosome 14 was located in the immunoglobulin heavy immunoglobulin indices [5]. The signal from chromosome 14 was located in the immunoglobulin chain locus near the immunoglobulin alpha 1 element (see Supplementary Table S4). Although the heavy chain locus near the immunoglobulin alpha 1 element (see Supplementary Table S4). Although most significant signal from chromosome 6 was highly correlated with HLA-DRB1*1501, this signal the most significant signal from chromosome 6 was highly correlated with HLA-DRB1*1501, this signal finds itself clearly distant from HLA genes. Similarly, the signal from chromosome 18 maps to the finds itself clearly distant from HLA genes. Similarly, the signal from chromosome 18 maps to the intergenic region between the genes ELAC1 (encoding the short form of RNase Z) and SMAD4 (C. intergenic region between the genes ELAC1 (encoding the short form of RNase Z) and SMAD4 elegans small body size (SMA) and Drosophila mothers against decapentaplegic (MAD) homolog 4). It (C. elegans small body size (SMA) and Drosophila mothers against decapentaplegic (MAD) homolog 4). seems likely that the identified markers represent tagging polymorphisms that are linked to yet It seems likely that the identified markers represent tagging polymorphisms that are linked to yet unknown functional elements. unknown functional elements. The analyses of the chromosomal regions surrounding the identified polymorphisms yielded The analyses of the chromosomal regions surrounding the identified polymorphisms yielded the presence of a variety of putative ERV sequences. In the surroundings of the SNP rs9807334 on the presence of a variety of putative ERV sequences. In the surroundings of the SNP rs9807334 on chromosome 18, the ORF (see Figure 1) that encodes a protease and reverse transcriptase has a very chromosome 18, the ORF (see Figure1) that encodes a protease and reverse transcriptase has a very high homology to a sequence identified in MSRV [8]. The corresponding original nucleotide sequence high homology to a sequence identified in MSRV [8]. The corresponding original nucleotide sequence (GenBank accession number AF009668) is not present in the human reference genome. It has been (GenBank accession number AF009668) is not present in the human reference genome. It has been suggested that MSRV sequences might arise by (probably post-transcriptional) recombination between different MSRV-like loci in the human genome [10]. The location of rs9807334 in the vicinity

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Int.suggested J. Mol. Sci. that 2018 MSRV, 19, x FOR sequences PEER REVIEW might arise by (probably post-transcriptional) recombination between5 of 7 different MSRV-like loci in the human genome [10]. The location of rs9807334 in the vicinity of ofa protease/polymerasea protease/polymerase open open reading reading frame frame with with such such high high homology homology to to the the isolatedisolated MSRVMSRV could supportsupport the the hypothesis hypothesis that that ERV ERV activity activity is isinvolved involved in inthe the pathogenesis pathogenesis of MS. of MS. TheThe SNPSNP on on chromosome chromosome 14 (located 14 (located in the immunoglobulinin the immunoglobulin heavy chain heavy locus) chain and chromosome locus) and 6 chromosome(located in the 6 (located MHC) mightin the MHC) be more might directly be more involved directly in involved the regulation in the regulation of the immune of the responseimmune responseand antibody and antibody production. production. Nevertheless, Nevertheless, these loci thes alsoe loci contain also contain retrovirus-like retrovirus-like open reading open reading frames frames(Figures (Figures3 and4). 3 The and investigated 4). The investigated region on regi chromosomeon on chromosome 14 contains 14 multiple contains ERV-like multiple sequences. ERV-like sequences.Of special interestOf special is the interest trio of is ERV-sequences the trio of ERV-sequences that are located that inare the located immunoglobulin in the immunoglobulin heavy chain heavylocus. Thesechain locus. ORFs includeThese ORFs the complete include the set ofcomplete sequences set (gag,of sequences pol, env) (gag, typically pol, foundenv) typically in retroviruses. found inThe retroviruses. location of The this location trio in theof this chromosomal trio in the chromosomal region containing region the containing immunoglobulin the immunoglobulin heavy chain heavyvariable chain (V) elementsvariable (V) (see elements Supplementary (see Supplem Tableentary S4) indicates Table S4) that, indicates during Bthat, cell during development, B cell development,immunoglobulin immunoglobulin rearrangements canrearrangements substantially reducecan substantially the distance reduce of these the elements distance from of the these SNP elementsrs11621145. from Immunoglobulin the SNP rs11621145. class switching Immunoglobulin can further class reduce switching this distance can further or can reduce lead tothis excision distance of orthe can rs11621145 lead to excision region. of the rs11621145 region.

Figure 4. Schematic organization of the rs9271640/rs6457617 region at human chromosome 6. The Figure 4. Schematic organization of the rs9271640/rs6457617 region at human chromosome 6. 2,071,651 base pair region on human chromosome 6 flanking the rs9271640/rs6457617 region was The 2,071,651 base pair region on human chromosome 6 ﬂanking the rs9271640/rs6457617 region analyzed for the presence of open reading frames (ORFs) with a minimal length of one kilobase by was analyzed for the presence of open reading frames (ORFs) with a minimal length of one kilobase by using getorf. 27 ORFs were identified (see Supplementary Table S5). These ORFs were analyzed using using getorf. 27 ORFs were identiﬁed (see Supplementary Table S5). These ORFs were analyzed using BLASTP against the database of retro-transcribing viruses (taxid 35268). One of the ORFs (ORF15) BLASTP against the database of retro-transcribing viruses (taxid 35268). One of the ORFs (ORF15) showed high homology to known sequences from retro-transcribing viruses. For a complete list of all showed high homology to known sequences from retro-transcribing viruses. For a complete list of genes in this chromosomal region, see Supplementary Table S6. TAP: transporter associated with all genes in this chromosomal region, see Supplementary Table S6. TAP: transporter associated with antigen processing. HLA: human leukocyte antigen. antigen processing. HLA: human leukocyte antigen.

The SNPs on chromosome 6 are located in the human major histocompatibility complex (MHC) class TheII region. SNPs The on chromosomeadjacent ORF 6 that are encodes located ina puta the humantive envelope major histocompatibilityprotein has high similarity complex to (MHC) other humanclass II region.ERVs. Nearly The adjacent identical ORF sequences that encodes have a putativebeen detected envelope on proteinother human has high chromosomes similarity to (EAW95690other human [11], ERVs. AAB65470, Nearly AAB65469 identical sequences [12]). In addition have been, the detected sequence on has other some human similarity chromosomes with other members(EAW95690 of [the11], K AAB65470, family including AAB65469 HERV-K113 [12]). In addition, and HERV-K18. the sequence The hasHERV-K113 some similarity sequence with is other not presentmembers in all of theindividuals K family [13]. including HERV-K113 HERV-K113 positivity and was HERV-K18. found in some The HERV-K113studies to be sequenceassociated is with not MSpresent and inother all individualsautoimmune [13 diseases]. HERV-K113 [14,15]. positivity However, was other found studies in some were studies unable to to be found associated this associationwith MS and [16]. other autoimmune diseases [14,15]. However, other studies were unable to found this associationThe interaction [16]. of different MS susceptibility loci is already known and may lead to considerable MS-risk amplification [17,18]. The question of how ERV elements interfere with OCB associated loci and result in production of intrathecal OCB IgG remains to be established. It is known that retroviral envelope proteins like gp120 of HIV activate B cells to produce antibodies [19]. In fact, at least 95% of the oligoclonal IgG synthesis within the CSF of HIV positive patients is not directed against HIV

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The interaction of different MS susceptibility loci is already known and may lead to considerable MS-risk ampliﬁcation [17,18]. The question of how ERV elements interfere with OCB associated loci and result in production of intrathecal OCB IgG remains to be established. It is known that retroviral envelope proteins like gp120 of HIV activate B cells to produce antibodies [19]. In fact, at least 95% of the oligoclonal IgG synthesis within the CSF of HIV positive patients is not directed against HIV antigens [20]. Therefore, it is not far to speculate that a major part of these antibodies may be due to B cell stimulation, e.g., by the HIV envelope protein gp120. From the putative ERV sequences detected in our study, only two (ORF87 from Supplementary Table S3 and ORF15 from Supplementary Table S5) have high sequence similarities to retroviral envelope proteins. In both cases, the detected ORFs encoded only the C terminal part of an envelope including the typical heptad repeat 1/2 ectodomain region. The ORFs encode approximately 69% (ORF87) and 49% (ORF15) of a typical envelope protein. Whether these sequences are translated into proteins have to be investigated in further studies. If such sequences are translated into functional envelope proteins, it might be possible that these factors stimulate B cells and therefore inﬂuence antibody production in these cells.

4. Materials and Methods SNPs to analyze were selected on the basis of a published study concerning associations between SNPs and CSF immunoglobulin levels in MS patients [5]. From this study, only SNPs were considered if they showed significant associations with CSF immunoglobulin levels in the screening phase as well as in the replication phase. Only 4 SNPs fulfilled this criterion: rs9807334 from chromosome 18, rs11621145 from chromosome 14, and the two SNPs rs9271640 and rs6457617 from chromosome 6. Analysis of the chromosomal regions surrounding these SNPs for the presence of putative ERV sequences was essentially performed as previously described [7]. Genomic SNP sequences were retrieved from the NCBI SNP data base (https://www.ncbi.nlm.nih.gov/projects/SNP/) and blasted against the current human reference genome (NC_000014.9) using NCBI nucleotide BLAST [21]. The two megabase pairs surrounding the SNPs were analyzed with getorf (http://bioinfo.nhri.org.tw/cgi-bin/emboss/ getorf)[22] for the presence of large (>999 nucleotides) open reading frames. The identified open reading frames were tested for sequence similarity to retro-transcribing viruses (taxid 35268) using BLASTP [21]. Gene annotations were retrieved from the reference genome sequence (chromosome 14: NC_000014.9).

5. Conclusions Taken together, retroviral elements exist in the relatively close vicinity of major genetic determinants for OCB and for IgG indices in MS. The precise meaning of how these ORFs with homology to retroviruses with proposed involvement in MS pathogenesis may come into play remains to be established.

Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/19/3/ 786/s1. Acknowledgments: This work was supported by a grant from the Wilhelm Roux Program of the Medical Faculty of the Martin Luther University Halle-Wittenberg (Alexander Emmer and Martin S. Staege). We gratefully acknowledge generous support from Novartis Pharma GmbH, Nuremberg, Germany. Author Contributions: Alexander Emmer, Christine Brütting and Martin S. Staege performed experiments. Alexander Emmer, Christine Brütting, Malte Kornhuber and Martin S. Staege analyzed the data and wrote the paper. Conﬂicts of Interest: The authors declare no conﬂicts of interest.

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