Med Linkage rheumatoid arthritis candidate NRAMP1 on 2q35

67 Med Genet 1996;33:672-677

Linkage of rheumatoid arthritis to the candidategene NRAMP1 on 2q35

Marie-Anne Shaw, David Clayton, Sara E Atkinson, Hazel Williams, Nancy Miller, DeanSibthorpe, Jenefer M Blackwell

AbstractThe macrophage resistance geneNRAMP1 regulates priming/activation ofmacrophages for enhanced TNFa, ILIl,and MHC class II expression. Since all ofthese functions are of potential im-portance in the induction or maintenanceor both of autoimmune disease, samplesfrom the Arthritis and RheumatismCouncil's repository of multicase rheu-matoid arthritis families were typed for adinucleotide repeat in the NRAMP1 pro-moter region and four other 2q34 (TNP1)or 2q35 (IL8R, VILl, DES) marker genes.Identity by descent (IBD) sib pair analysisusing a three locus haplotype NRAMP1-IL8RB-VIL1, or NRAMP1 alone, providedpreliminary evidence (maximum lodscore = 1.01, p = 0.024) for a gene in thisregion contributing to susceptibility torheumatoid arthritis. Candidacy forNRAMP1 as the disease susceptibilitygene was supported by a significant bias(p=0.048) towards transmission of theNRAMP1 promoter region allele 3 inaffected offspring.(JMed Genet 1996;33:672-677)

Key words: linkage; rheumatoid arthritis; NRAMP1.

Department ofMedicine, Universityof Cambridge ClinicalSchool, Level 5,Addenbrooke'sHospital, Hills Road,Cambridge CB2 2QQ,UKM-A ShawS E AtkinsonH WilliamsN MillerD SibthorpeJ M Blackwell

Medical ReseachCouncil BiostatisticsUnit, Institute ofPublic Health,University Forvie Site,Robinson Way,Cambridge CB2 2SR,UKD Clayton

Correspondence to:Dr Shaw or ProfessorBlackwell.

Received 30 November 1995Revised version accepted forpublication 22 April 1996

Two questions of fundamental importance inrheumatoid arthritis are (1) which genes conferdisease susceptibility,' and (2) does an infec-tious agent trigger disease?2 Epidemiologicaldata suggest a two locus multiplicative modelfor genetic susceptibility,3 to which HLA-DRhas been estimated to contribute 37%.' Themajor gene(s) accounting for the remaining63% has yet to be identified.A plausible candidate for non-MHC gene

regulation of early induction of rheumatoidarthritis is the human homologue, NRAMP1,for the murine macrophage resistance gene ItylLsh/Bcg (Nrampl). Ity/Lsh/Bcg was identified as

a single gene controlling recessive susceptibilityto the intramacrophage pathogens Salmonellatyphimurium, Leishmania donovani, and Myco-bacterium bovis BCG, and was mapped to thedistal end of mouse chromosome 1.5-7 A candi-date for ItylLshlBcg was identified by positionalcloning,8 a full length cDNA sequence ob-tained,9 and its candidacy confirmed byfunctional analysis of transfected macrophagecell lines. ' The gene has been renamedNrampl, the Natural resistance associatedmacrophage protein.8 Through a region ofconserved synteny on chromosome 2q35,

human NRAMP1 has been sequenced" anddetailed physical and genetic mapping of theregion undertaken.'2 This work also showedthat the IL8 receptor gene cluster, IL8RA,IL8RB, and the pseudogene IL8RBP, lies inclose proximity (-130 kb distal) to theNRAMP1 gene.

Functional studies indicate that Nramplregulates, and acts early in the pathway to,macrophage priming/activation for antimicro-bial activity.5-7 The gene therefore has manypleiotropic effects on macrophage function,including differential upregulation of the earlyresponse gene KC, a neutrophil chemoattract-ant belonging to the C-X-C family of smallcytokines, as well as TNFa, IL1,B, induciblenitric oxide synthase, and MHC class IIexpression, leading to higher levels in resistantmacrophages. All of these functions are ofpotential importance in the induction or main-tenance, or both, of autoimmune disease.Hence, an animal resistant to intracellularpathogens may be susceptible to autoimmunedisease. A number of agents may triggerNrampl regulated macrophage responses, in-cluding interferon y, bacterial lipopolysaccha-ride (LPS), and mycobacterial lipoarabi-nomannan (LAM). A role for NRAMP1 inrheumatoid arthritis would therefore also beconsistent with evidence for a bacterial/mycobacterial aetiology,' '3 either as a triggerfor autoimmunity or as an agent for theperpetuation of disease.To test the hypothesis that NRAMP1

influences rheumatoid arthritis, samples fromthe Arthritis and Rheumatism Council (ARC)Repository were typed for a dinucleotiderepeat in the NRAMP1 promoter region" andfor polymorhisms at four other 2q34/35 genes,and the data analysed using an affected sib pairmaximum likelihood identity by descent (IBD)method of linkage analysis and transmissiondisequilibrium testing.

Material and methodsFamilies from the ARC National Repositoryhave been described in detail by Worthingtonet al'4 and Hay et al'5 including diagnostic crite-ria and family structures. In this study, 61nuclear families from the repository were ana-lysed, described in Worthington et al'4 as fami-lies 001-046,051-066,075-109, 129-182,196,198-201, 205-210, 237, 238, 309, and 319.Five of these families had three affectedoffspring and one family had four affected off-spring, giving a total of 76 affected sib pairs(although a weighting factor was applied in sib

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Linkage of rheumatoid arthritis to NRAMP on 2q35

Table 1 Pairwise lod scores (sex averaged) for linkage between pairs ofgenes on 2q33-q35from 61 pedigrees analysed using LIPED

Recombination fraction

0 0.001 0.01 0.05 0.1 0.2 0.3 0.4

TNPl-NRAMPI - 98.78 - 2.28 - 0.32 0.95 1.34 1.29 0.81 0.26NRAMP1-IL8R 3.87 3.87 3.80 3.48 3.02 2.05 1.09 0.31IL8R-VIL1 4.04 4.03 3.93 3.47 2.90 1.82 0.91 0.25VILI-DES - 94.55 3.03 3.94 4.16 3.81 2.71 1.51 0.50

pair analyses for families with multiple sibs,reducing the effective number of sib pairs con-

tributing to the analysis to 68, see below).Nineteen sib pairs had both parents typed, 10sib pairs had one parent typed, and 32 had no

parental DNA available for typing. The totalnumber of DNA samples available for typingfor 2q34/35 genetic polymorphisms was 318,129 affected and 189 unaffected.Samples from all family members were

included in typing for a repeat polymorphismimmediately 5' of position -594 bp in theNRAMP 1 promoter region." Four variantsoccur within a possible enhancer element con-

taining the Z-DNA forming dinucleotiderepeat originally sequenced as

t(gt)5ac(gt)5ac(gt)9g. Alleles 1, 2, and 3 vary inrepeat number (11, 10, 9 repeats, respectively)for the larger array of gt repeats. Allele 4 carriesthe sequence t(gt)5ac(gt)l g. All samples were

also included in typing for a new restrictionfragment length polymorphism (RFLP) inIL8RB,'2 and for RFLPs in genes proximal(TNP1, 2q34) and distal (VILl and DES,2q35) to NRAMP1 and IL8R.'6 The gene

order is known to be TNP1-NRAMP1-IL8RA-IL8RB-VIL1-DES. The majority ofsamples provided a scorable genotype. Linkagebetween these genes was analysed by LIPED,'7using previously reported'2 16 gene frequencies:TNP1 A1=0.366 A2=0.634; IL8R A1=0.314A2=0.686; VILl A1=0.672 A2=0.328; andDES A1=0.659 A2=0.341. NRAMP1 allelefrequencies were those calculated by SPLINK(see below) in this analysis: A1=0.001A2=0.246 A3=0.753 A4=0.001.

Linkage between markers and disease sus-

ceptibility was analysed by the affected sib pairmethod, using the maximum likelihood iden-tity by descent (IBD) method pioneered byRisch'8 19 and later extended by Holmans20 andHolmans and Clayton2' to allow for uncer-

tainty of IBD assignment owing to unknownphase of marker haplotypes and to missing or

incomplete parental genotype data. The statis-

tical method involves the use ofmaximum like-lihood to estimate the IBD sharing probabili-ties and haplotype frequencies simultaneously,and is implemented in the computer programSPLINK (available from the second author).In the IBD analyses, affected sib trios weredealt with by treating each trio as the threeindependent pairwise comparisons, but givingeach comparison a weight of only 2/3 in theanalysis.22 Similarly the six possible pairwisecomparisons of the affected 4-tuple were eachgiven a weight of 1/2. This is a conservativestrategy (P Holmans, D Clayton, unpublishedobservation). For each marker locus examined,the transmission of individual alleles from het-erozygous parents to affected offspring was

examined using the transmission disequilib-rium test.23 24

ResultsUSE OF A THREE LOCUS HAPLOTYPEMaximum lod scores for linkage betweenNRAMP1-IL8R and IL8R-VIL1 occurred at a

recombination fraction of 0 (table 1), indicat-ing that there were no recombinants betweenthese markers in the 61 rheumatoid arthritisfamilies. A haplotype for these three loci was

therefore used in the sib pair analyses, increas-ing the heterozygosity from 0.371 (NRAMP1alone) to 0.766. Zero recombination betweenthese markers is consistent with the physicalNRAMP1-VIL1 interval of 155 kb and IL8R-VILl interval of 10-30 kb.'2

SIB PAIR ANALYSISThe results of the IBD analyses for all markersare summarised in table 2. IBD sharingprobabilities (0:1:2 alleles IBD) for the threelocus haplotype, estimated using the maximumlikelihood method,'8 19 were 0.118:0.500:0.382giving a likelihood ratio chi-squared test statis-tic of 4.63. This provides a maximum lod score

(MLS) of 1.01 which corresponds asymptoti-cally to statistical significance at the p=0.024level.20 The contributions which individualfamilies make to these probabilities are repre-sented diagramatically in fig 1. Affected sibpairs whose IBD status is known exactly plot atthe vertices of the triangles. The plotting posi-tions of pairs calculated using maximum likeli-hoods reflect the relative probabilities of theirassignments. In the left hand figure, theplotting positions for the probabilistic IBDassignments assume the null hypothesis (nolinkage, 0.25:0.5:0.25). This plot represents

Table 2 Summary ofIBD sib pair analysis using SPLINK After weighting, the 76 affected sib pairs equated to 65-68independent sib pairs contributing to the analysis for different genes. Effective sample size is calculated by SPLINK as theequivalent number offully informative sib pairs. X2 = likelihood ratio chi-squared test statistic. MLS = maximum lod score,p = probability, PIC = polymorphic information content

Estimated IBD sharing probabilitiesEffective

0-ibd l-ibd 2-ibd sample size x2 MLS p

TNP1 0.250 0.500 0.250 19 0 0 0NRAMP1 0.127 0.420 0.453 13 4.64 1.01 0.024NRAMP1-IL8R-VIL1 0.118 0.500 0.382 27 4.63 1.01 0.024DES 0.182 0.500 0.318 19 0.90 0.20 0.171

NRAMP1: heterozygosity = 0.371, PIC score = 0.302.NRAMPl-IL8R-VILI: heterozygosity = 0.766, PIC score = 0.740.TNP1: heterozygosity = 0.487, PIC score = 0.368.DES: heterozygosity = 0.437, PIC score = 0.342.

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1 IBD(4)

!7

0 IBD

1 IBD(4)

Null hypothesis

(5)2 IBD

Maximum likelihood

Figure 1 Contributions of individual families to IBD haplotype sharing probabilities. An affected sib pair whose IBDstatus is known exactly is plotted at the vertices of the triangles. The plotting position of other pairs depends on the relativeprobabilities of their assignments. All 59 families informative for the three locus haplotype contribute to the maximum lodscore. Open circles indicate single sib pairs while closed circles indicate the contribution of more than one sib pair. In thelatter case the number ofpairs is shown alongside the plotting point.

Table 3 NRAMPI-IL8R-VILI haplotype frequenciesderivedfrom 59families/66 sib pair comparisons usingSPLINK

Haplotype Frequency Haplotype Frequency

1-1-1 < 0.001 1-1-2 < 0.0012-1-1 0.010 2-1-3 0.0173-1-1 0.133 3-1-2 0.0771-2-1 < 0.001 1-2-2 < 0.0012-2-1 0.134 2-2-2 0.0833-2-1 0.406 3-2-2 0.138

the evidence on which the significance test isbased. The right hand figure uses the assign-ment probabilities at the final maximum likeli-hood estimate of the sharing probabilities.Summation of these assignments provides themaximum likelihood estimates. None of the 10fully informative families showed 0-IBD for theNRAMP1-IL8R-VIL1 haplotype. The posi-tions for the 49 families obtained usingprobability assignments depict the clear bal-ance of probability away from the null hypoth-esis. In this maximum likelihood analysis, themarker haplotype frequencies are estimatedinternally. This guards against errors whichmay follow from misspecification by takinghaplotype frequencies from inappropriatepopulations. The fact that these frequenciesare estimates rather than known constants isallowed for in the likelihood ratio tests. Thefinal estimates of the NRAMP1-IL8R-VIL1haplotype frequencies are shown in table 3.

Table 4 Transmission disequilibrium testing

No of transmissions from No of transmissionsheterozygous parents No offamilies Allele for each allele

TNP1 54 38 1 222 32

NRAMP1 31 25 2 10X2=3.93 21p=0.048

IL8R 21 15 1 122 9

VILl 38 30 1 192 19

DES 52 35 1 212 31

When the IBD analysis was repeated forNRAMP1 alone (table 2), the maximum lodestimates of the IBD sharing probabilities were(0.127:0.420:0.453), again giving a likelihoodratio chi-squared test statistic of 4.64 and aMLS of 1.01. Hence, even without theadditional polymorphic information contentwhich the haplotype provides, a positiveassociation between NRAMP1 and suscepti-bility to rheumatoid arthritis is observed. Incontrast, no linkage between rheumatoid ar-thritis and the markers either proximal(TNP1) or distal (DES) to the three locushaplotype (table 2) was observed, even thoughthe polymorphic information content for thesemarkers was higher than for the NRAMP1promoter region polymorphism.

TRANSMISSION TESTINGWhile NRAMP1 is a candidate locus, and link-age has been established to 2q35, it is possiblethat another gene in the region of the threelocus haplotype is responsible for susceptibil-ity. To address this issue, transmission of allelesfrom heterozygous parents was examined forthe five 2q34/35 genes using the same set offamilies (table 4). Since problems may arise intransmission testing where parental genotypeinformation is incomplete,25 only matingswhere parental genotypes were conclusivelyassigned were used in the analysis. In themajority of cases this meant families whereboth parents were available for typing. ForNRAMP1, two families were included wherethe heterozygous parental genotype was de-duced on the basis of genotypes of offspringfrom the extended family, that is, unaffected aswell as affected offspring were available. Inboth cases, there was only one possible paren-tal genotype which could be assigned, and thiscould be done without reference to the affectedperson being tested for transmission disequi-librium. The only significant deviation fromthe expected 1:1 transmission of the two allelesto affected sibs under the null hypothesis wasobtained for NRAMP1, where allele 3 was

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Table 5 Risk of rheumatoid arthritis in sib sharing 0, 1, or2 NRAMP-IL8R-VIL1 2q35 or HLA haplotypes IBDwith an affected sib

NRAMP1-IL8R-VILI HLA(%/°) (%/)

0-IBD 1.8 2.31-IBD 3.9 3.52-IBD 6.0 6.2

transmitted to affected children in preferenceto allele 2 (chi-squared=3.9, p=0.048). In test-ing for transmission disequilibrium, multipleindependent tests have been carried out.Hence, it could be argued that a multiple test-ing correction factor should be applied. Multi-plying our p values by the number of tests per-formed (2 alleles x 5 loci = 10) would clearlyremove all significance from the NRAMP 1

TDT allelic association in this small dataset.However, as Copeman et alf6 argue, if we hadtested only the loci showing independentevidence of linkage (NRAMP1), a correctionfactor of only 2 (= 2 alleles, 1 locus) could beapplied. Since we could also argue that a one

tailed test could be applied in comparingalleles 2 versus 3, that is, that we would expecta one way skew towards allele 3 in affected off-spring on functional grounds (S Searle, J MBlackwell, unpublished data, see Discussion),our original p value for NRAMP 1 TDT couldbe halved. Multiplying by 2 would then retainthe TDT for NRAMP1 within the 5% signifi-cance level.

RELATIVE IMPORTANCE OF A GENE AT 2Q35Using the sib recurrence risk of 3.9%,4 the riskof disease to sibs sharing 0, 1, or 2 NRAMP1-IL8R-VIL1 haplotypes IBD with an affectedsib were calculated and compared with previ-ous estimates" for HLA haplotype sharing(table 5). The value of 6.0% for a sib sharingtwo NRAMP1 haplotypes IBD is comparableto the risk of 6.2% calculated for HLA."7 Thevalue of 2.3% for 0-IBD HIA haplotypes,which is greater than the observed populationprevalence of 1 %28 for sibs sharing 0-IBD HLAhaplotypes, is itself indicative of other factorsoperating, such as additional genetic loci. Thedegree of familial clustering is the ratio of therisk to the sibs of affecteds and the populationprevalence: = 3.9 for rheumatoid arthritis.29The relative contribution of a locus to this totalis the ratio of expected to observed sharing no

alleles IBD"6: 0.25/0.118 = 2.1 (a) for theNRAMP1-IL8R-VIL1 haplotype, 0.25/0.127= 2.0 for NRAMPPI alone, and 0.25/0.15 = 1.7(b). for HLA. Using the additive model ofRisch,30 where either NRAMP1 or HLA haplo-types could confer susceptibility, a totalcontribution [(a-i) + (b-i) + 1] of 2.8(1.1 + 0.7 + 1) is obtained. A multiplicativemodel (a x b), where both loci operate, gives atotal contribution of 3.6 (2.1 x 1.7). Whicheveris the case, it is clear that NRAMP1 (or a

tightly linked gene) could make a significantcontribution to disease susceptibility, compa-rable to that observed previously for HLA.

DiscussionResults of the IBD sib pair analysis presentedhere provide evidence (MLS=1.01, p=0.024)for the presence of a gene contributing to sus-ceptibility to rheumatoid arthritis in the regionof the NRAMP1-IL8R-VIL1 haplotype onhuman chromosome 2q35. Since this regioncontained an a priori candidate gene(NRAMP1) for disease susceptibility on func-tional grounds, there was no multiple testingproblem. We have performed a single test ofthe null hypothesis of no linkage to 2q35, witha resulting pointwise nominal significancelevel31 of 2.4%. Since this region was not iden-tified as part of a genome wide search, themore stringent criteria for genome widesignificance levels suggested by Lander andKruglyak3' do not apply. Candidacy forNRAMP1 as the disease susceptibility genewithin 2q35 was supported by (1) IBD analysisfor NRAMP1 alone providing an equivalentMLS of 1.01, and (2) a significant bias(p=0.048) towards transmission of theNRAMP1 promoter region allele 3 in affectedoffspring. The latter observation suggests thatthe NRAMP1 promoter region polymorphismitself may be the disease associated geneticdefect in these families. Although a role forIL8R genes cannot be discounted, NRAMP 1itself does provide the more plausible func-tional candidate for non-MHC control of sus-ceptibility to rheumatoid arthritis. Its pleio-tropic effects5-7 include roles in cytokineproduction, in particular TNFa and ILI f, andin MHC class II antigen presentation to CD4+T cells. There is evidence that macrophages aredirectly involved in the inflammatory processin rheumatoid arthritis, and that TNFx plays amajor role in autoimmune disease. ILl, andTNFak are both expressed by synovial tissuemacrophages in rheumatoid arthritis.32 GM-CSF is also produced in large quantities byactivated macrophages in the synovium, andacts to induce IL- 1,B production and enhanceclass II expression.33 Hence, there is goodevidence that the activation phenotypes associ-ated with NRAMP1 regulated macrophageactivation could contribute directly to theinflammatory response and pathology associ-ated with rheumatoid arthritis. Results of con-tinuing research in our laboratory (S Searle, JM Blackwell, unpublished data) analysing pro-moter function in a reporter gene system indi-cate that allele 3 of the NRAMP 1 promoterregion polymorphism drives NRAMP1 expres-sion more efficiently than allele 2. This wouldbe consistent with overexpression of theNRAMP1 gene, and hence hyperactivation ofmacrophages, contributing to disease suscepti-bility. Although additional functionalpolymorphisms/mutations in NRAMP1 maybe found, there is good evidence that this pro-moter region polymorphism may represent thefunctional 2q35 polymorphism contributing todisease susceptibility in families analysed here.

In previous studies, polymorphic HLA hap-lotypes have been shown to contribute todisease susceptibility in rheumatoid arthritis,either through the direct role of MHC class IImolecules in presenting antigen to autoim-

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mune T cells or through linkage disequilibriumwith polymorphic elements regulating TNFoxproduction.34 The possible interaction betweenNRAMP1 and MHC class II regulation of dis-ease susceptibility is of particular interest. Onehypothesis for polygenic control in rheumatoidarthritis is that non-MHC genes determinesusceptibility to disease per se and act early inthe induction phase of autoimmunity, whereasHLA determines disease severity throughmodulation of T cell responses to particularantigens.35 36 This parallels observations in themouse where Nrampl influences the earlymacrophage response to infection, and hencesusceptibility to disease per se, while polymor-phism in class II molecules at the mouse H-2locus influences disease severity.37 For controlof infection, the best possible interactionbetween genes occurs when an Nrampl resist-ant mouse carries an H-2 curing haplotype,leading to enhanced antigen presentation toprotective interferon y generating CD4+ Thelper 1 cells. In autoimmune disease, thismight represent the worst possible interaction.T cells, particularly CD4 + cells, contribute topathology in rheumatoid arthritis. The involve-ment of cells recognising antigen in associationwith HLA class II molecules fits well with theassociation of rheumatoid arthritis with par-ticular DR types36 and also points to asignificant role for antigen presenting cellssuch as macrophages. Further analysis of theinteraction between DR and NRAMP1 indetermining RA susceptibility will be ofparticular interest.NRAMP 1 as a candidate for susceptibility to

rheumatoid arthritis is also interesting inrelation to a bacterial/mycobacterial aetiologyfor the disease. The gene was first identified forits role in controlling resistance to intra-macrophage pathogens. Many arthritic dis-eases have been shown to have intracellularpathogens as triggers, and the hypothesis thatrheumatoid arthritis is a slow bacterial infec-tion with autoimmunity a secondary conse-quence has not been disproved.'3 In the case ofrheumatoid arthritis, no self antigen has beenshown to be associated with the initiation ofdisease. Mycobacteria contain inducers ofTNF-oc, the most potent of which is LAM,38while organisms associated with reactive ar-thritis have LPS which can be identified inaffected joints.39 Both LAM and LPS areimportant triggers ofNrampl regulated macro-phage activation.6 7 Support for a role of myco-bacteria in arthritis has come from the use ofLewis rats which are susceptible to adjuvantinduced arthritis and respond particularly tothe mycobacterial 65 kDa hsp. Indeed, arthritisis inducible by a single T cell clone recognisingthe non-conserved 180-188 region of myco-bacterial hsp65.4° T cells isolated from thesynovial fluid of patients have been found togive a localised response to both conserved andnon-conserved mycobacterial hsp65 epitopes."The reactivity against non-conserved epitopesof hsp65 cannot be accounted for by crossreactions with the human homologue. Again,the interaction between NRAMP1 and MHCclass II molecules in directing the T cell

response to bacterial/mycobacterial antigensmay be of particular importance in initiationand maintenance of disease.The primary function of NRAMP1 remains

unknown. While its molecular structure iscompatible with a transport function, the pres-ence of an SH3 binding domain at theN-terminus of the molecule points to a role insignal transduction.9 In the murine system, thefinal effector mechanism for antimicrobialactivity relies on induction of nitric oxide syn-thase and generation of nitric oxide.7 This, orany one or combination of the multiplepleiotropic effects of the gene, would besufficient to account for a role for NRAMP 1 inregulating autoimmune disease.

In this study, only 47% of families had one(16%) or both (31%) parents of affected sibsavailable for study, reducing the power of ouranalysis in terms of equivalence to fullyinformative affected sib pairs (table 2). This isa general problem in late onset diseases. Ourresults are therefore preliminary, and requireconfirmation in additional datasets before thereal contribution of NRAMP1 to diseasesusceptibility in rheumatoid arthritis is known.Functional studies analysing NRAMP1 ex-pression in synovial tissue macrophages wouldalso be of interest, as well as studies comparingactivation phenotypes in macrophages frompeople bearing different NRAMP1 genotypes.These continuing studies of NRAMP1 inrheumatoid arthritis patients and families pro-vide an exciting basis to further genetic andfunctional characterisation of disease suscepti-bility.

This work was supported by grants from the Arthritis andRheumatism Council and the Wellcome Trust. We would like tothank Dr Bill Ollier, ARC Epidemiology Research Unit,Manchester University, for his help in making material availablefrom the ARC Repository.

1 Wordsworth P, Bell J. Polygenic susceptibility in rheumatoidarthritis. Ann Rheum Dis 1991;50:343-6.

2 Silman AJ. Is rheumatoid arthritis an infectious disease?BMJ 1991;303:200.

3 Rigby AS, Voelm L, Silman AJ. Epistatic modelling in rheu-matoid arthritis: an application of the Risch theory. GenetEpidemiol 1993;10:311-20.

4 Deighton CM, Walker DJ, Griffiths ID, Roberts DF. Thecontribution of HLA to rheumatoid arthritis. Clin Genet1989;36: 178-82.

5 Blackwell JM (Convenor, 27th Forum in Immunology). Themacrophage resistance gene Lsh/Ity/Bcg. Res Immunol1989;140:767-828.

6 Blackwell JM, Roach TIA, Atkinson SE, et al. Genetic regu-lation of macrophage priming/activation: the Lsh genestory. Immunol Lett 1991;30:241-8.

7 Blackwell JM, Barton CH, White JK, et al. Geneticregulation of leishmanial and mycobacterial infections: theLshlItylBcg gene story continues. Immunol Lett 1994;43:99-107.

8 Vidal SM, Malo D, Vogan K, Skamene E, Gros P. Naturalresistance to infection with intracellular parasites: isolationof a candidate for Bcg. Cell 1993;73:469-85.

9 Barton CH, White JK, Roach TIA, Blackwell JM.NH2-terminal sequence of macrophage-expressed naturalresistance-associated macrophage protein (Nramp) en-codes a proline/serine-rich putative Src homology3-binding domain. Jf Exp Med 1994; 179: 1683-7.

10 Barton CH, Whitehead SH, Blackwell JM. Nramp transfec-tion transfers ItylLshlBcg-related pleiotropic effects onmacrophage activation: influence on oxidative burst andnitric oxide pathways. Mol Med 1995;1:267-79.

11 Blackwell JM, Barton CH, White JK, et al. Genomic organi-zation and sequence of the human NRAMP gene: identifi-cation and mapping of a promoter region polymorphism.MolMed 1995;1:194-205.

12 White JK, Shaw MA, Barton CH, et al. Genetic and physi-cal mapping of 2q35 in the region ofNRAMP and IL8Rgenes: identification of a polymorphic repeat in exon 2 ofNRAMP. Genomics 1994;24:295-302.

13 Rook G, McCulloch J. HLA-DR4, mycobacteria, heat-shock proteins, and rheumatoid arthritis. Arthritis Rheum1992;35: 1409-12.

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14 Worthington J, Ollier WER, Leach MK, et al. The Arthritisand Rheumatism Council's National repository of familymaterial: pedigrees from the first 100 rheumatoid arthritisfamilies containing affected sibling pairs. Br .7 Rheumatol1994;33:970-6.

15 Hay EM, Ollier WER, Silman AJ. The Arthritis and Rheu-matism Council's national family material repository. Br]Rheumatol 1993;32:443-4.

16 Shaw MA, Atkinson S, Dockrell H, et al. An RFLP map for2q33-q37 from multicase mycobacterial and leishmanialdisease families: no evidence for an LshlItylBcg genehomologue influencing susceptibility to leprosy. Ann HumGenet 1993;57:251-71.

17 Ott J. Linkage analysis and family classification underheterogeneity. Ann Hum Genet 1983;47:311-20.

18 Risch N. Linkage strategies for genetically complex traits. II.The power of affected relative pairs. Am . Hum Genet1990;46:229-41.

19 Risch N. Linkage strategies for genetically complex traits.III. The effect of marker polymorphism on analysis ofaffected relative pairs. Am _Hum Genet 1990;46:242-53.

20 Holmans P. Asymptotic properties of affected-sib-pair link-age analysis. Am Hum Genet 1993;52:362-74.

21 Holmans P, Clayton D. Efficiency of typing unaffected rela-tives in an affected sib-pair linkage study with single locusand multiple tightly-linked markers. Am 7 Hum Genet1995; 37:1221-32.

22 Payami H, Thomson G, Motro U, Louis EJ, Hudes E. Theaffected sib pair method. IV. Affected sib trios. Ann HumGenet 1985;49:303-14.

23 Spielman RS, McGinnis RE, Ewens WJ. Transmission testfor linkage disequilibrium: the insulin gene region andinsulin-dependent diabetes mellitus (IDDM). Am . HumGenet 1993;52:506-16.

24 Schaid DJ, Sommer SS. Comparison of statistics forcandidate-gene association studies using cases and par-ents. Am _Hum Genet 1994;55:402-9.

25 Curtis D, Sham PC. A note on the application of the trans-mission disequilibrium test when a parent is missing. Am]Hum Genet 1995;56:811-12.

26 Copeman JB, Cucca C, Hearne CM, et al. Linkage disequi-librium mapping of a type 1 diabetes susceptibility gene(IDDM7) to chromosome 2q31-q33. Nature Genet1995;9:80-5.

27 Rigby AS. HLA haplotype sharing in rheumatoid arthritissibships: risk estimates in siblings. Scand _7 Rheumatol1992;21:68-73.

28 Hazes JMW, Silman AJ. Review of UK data on rheumaticdiseases-2. Rheumatoid arthritis. Br ]7 Rheumatol 1990;29:310-12.

29 Risch N. Assessing the role of HLA-linked and unlinkeddeterminants of disease. Am _Hum Genet 1987;40:1-14.

30 Risch N. Linkage strategies for genetically complex traits. I.Multilocus models. Am _Hum Genet 1990;46:222-8.

31 Lander E, Kruglyak L. Genetic dissection of complex traits:guidelines for interpreting and reporting linkage results.Nature Genet 1995;11:241-7.

32 MacNaul KL, Hutchinson NI, Parsons JN, Bayne EK,Tocci MJ. Analysis of IL-1 and TNF-alpha geneexpression in human rheumatoid synoviocytes and normalmonocytes by in situ hybridization. .7 Immunol 1990;145:4154-66.

33 Alvaro-Gracia JM, Zvaifler NJ, Firestein GS. Cytokines inchronic inflammatory arthritis. IV Gm-CSF mediatedinduction of class II MHC antigen on human monocytes:a possible role in rheumatoid arthritis. ] Exp Med1989;170:865-76.

34 Jacob CO. Tumour necrosis factor-alpha in autoimmunity:pretty girl or old witch? Immunol Today 1992;13:122-5.

35 Wicks I, McColl G, Harrison L. New perspectives on rheu-matoid arthritis. Immunol Today 1994;15:553-6.

36 Singal DP, Green D, Reid B, Gladman DD, Buchanan WW,HLA-D region genes and rheumatoid arthritis (RA):importance of DR and DQ genes in conferring susceptibil-ity to RA. Ann Rheum Dis 1992;51:23-8.

37 Blackwell J, Freeman J, Bradley D. Influence of H-2complex on acquired resistance to Leishmania donovaniinfection in mice. Nature 1980;283:72-4.

38 Chatterjee D, Roberts AD, Lowell K, Brennan PJ, OrmeIM. Structural basis of capacity of lipoarabinomannan toinduce secretion of tumor necrosis factor. Infect Immun1992;60: 1249-53.

39 Granfors K, Jalkanen S, Linberg AA, et al. Salmonellalipopolysaccharide in synovial cells from patients withreactive arthritis. Lancet 1990;335:685-8.

40 Van Eden W, Thole JE, van der Zee R, et al. Cloning of themycobacterial epitope recognized by T lymphocytes inadjuvant induced arthritis. Nature 1988;331:171-3.

41 Gaston JS, Life PF, Jenner PJ, Colston MJ, Bacon PA. Rec-ognition of a mycobacteria specific epitope in the 65-kDheat-shock protein by synovial fluid-derived T cell clones.] Exp Med 1990;171:831-41.

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