Genome scan stratified by the presence of anti-double-stranded DNA (dsDNA) autoantibody in pedigrees multiplex for systemic lupus erythematosus (SLE) establishes linkages at 19p13.2 (SLED1) and 18q21.1 (SLED2)

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Anti-double-stranded DNA (anti-dsDNA) is arguably one of the most specific autoantibodies in systemic lupus erythematosus (SLE). This antibody is associated with more severe SLE and with glomerulonephritis. From 196 pedigrees multiplex for SLE, we selected those that had any SLE affected positive for anti-dsDNA by the Crithidia luciliae kinetoplast imunofluorescence assay. This stratification strategy tested the hypothesis that anti-dsDNA would identify a more genetically homogeneous group of pedigrees, in which previously undetected linkage effects could be established. A genome screen data for linkage to SLE was available at 307 microsatellite markers for this selected group of 71 pedigrees: 37 European-American, 29 African-American, and five others. The most significant results were obtained at 19p13.2 (LODmax = 4.93), named SLED1, in the 37 European-American pedigrees using a dominant model with mixed penetrances (92% for females and 49% for males) at 100% homogeneity (θ = 0). A second linkage effect, SLED2, was established in the 29 African-American pedigrees at 18q21.1 (LODmax = 3.40) using a recessive model with 100% penetrance (θ = 0.1). Parametric and non-parametric multipoint analyses were performed, which provided further evidence and support of susceptibility genes residing in these regions. In conclusion, two powerful linkages have been detected with SLE based on the presence of anti-dsDNA. These findings show SLE to be a richly complicated disease phenotype that is now ripe for important new discovery through a genetic approach.


Systemic lupus erythematosus (SLE) is an extraordinarily heterogeneous autoimmune disease with a strong genetic basis. Familial aggregation, high sibling recurrence rates (λs = 10 to 40),1 increased identical twin concordance,2 and the discovery of genetic linkages and associations all support there being susceptibility genes for SLE. Indeed, examples of genetic association include alleles at multiple genes in the HLA region, including HLA-DR3 and different alleles of complement components, and in the Fcγ region of 1q22-23.1.3,4 More recent work has also established multiple genetic linkages with SLE including effects at 1q21.3, 1q41, 2q37, 4p16, 6p21, and 16q13.5,6,7

The central immunologic disturbance in SLE is autoantibody production. These antibodies bind DNA, RNA, nuclear proteins and protein-nucleic acid complexes. Of the autoantibodies virtually characteristic of SLE (anti-double-stranded DNA (anti-dsDNA), anti-Sm and anti-P), only fluctuations in anti-dsDNA antibody levels are also used as markers for disease activity and an increase in the anti-dsDNA level may herald disease exacerbations.8 Despite this, anti-dsDNA usually can be detected even a few years before onset of SLE or severe disease flares.9 Different isotypes of anti-dsDNA antibodies may have different clinical consequences. For example, IgA anti-dsDNA has been associated with vasculitis10 and IgM anti-dsDNA has been negatively associated with lupus nephritis.11 The most specific feature of anti-dsDNA is the association of IgG (and especially IgG3) anti-dsDNA with glomerulonephritis in SLE.12,13

Many methods are being used to detect anti-dsDNA antibodies including solid phase assays (ELISA), precipitation assays (Farr assay), and immunofluorescence assays with Crithidia luciliae being the standard substrate. Based upon its high sensitivity and specificity, the C. luciliae immunofluorescence test is still considered the best assay for the detection of these autoantibodies. In fact, many of the commercial ELISA test systems often detect anti-single-stranded DNA antibodies, which are not specific for SLE and therefore may give false positive or negative results.14

We hypothesized that stratification of multiplex SLE pedigrees based on the presence of anti-dsDNA, as detected by the C. luciliae immunofluorescence method, may concentrate genetic effects because of this shared clinical feature. This clinical subdivision would then lead to increased genetic homogeneity and, therefore, more easily reveal genetic linkages in the selected pedigrees.


From a total of 196 multiplex SLE pedigrees, we selected all families that contained at least one affected with a positive anti-dsDNA titer, as detected by the C. luciliae immunofluorescence method (positive 1:10 dilution) in our clinical immunology lab (Figure 1). There were 71 pedigrees (37 European-American, 29 African-American, and five others) that satisfied this selection criterion. We compared their clinical and serological manifestations with the group of SLE pedigrees that consistently had no anti-dsDNA in any SLE affected, regardless of the method used (43 pedigrees: 33 European-American, six African-American and four others). We also performed linkage analysis in the selected pedigrees.

Figure 1

Examples of SLE pedigree diagrams for European-American, (EA) and African-American (AA) pedigrees with a positive anti-dsDNA.

Clinical and serological features

Frequencies of the 11 criteria for SLE classification15,16, as well as other associated clinical and serological manifestations, were examined in our selected group and were compared to the SLE patients from the pedigrees in which all of the patients consistently had a negative result for anti-dsDNA regardless of the methods used. In order to avoid dependent data by using multiple family members, we randomly selected one affected with a positive anti-dsDNA from each of our 71 selected pedigrees and compared them with one affected from 43 pedigrees who were anti-dsDNA negative (Table 1).

Table 1 Features of SLE patients with anti-dsDNA compared to SLE patients from families with no anti-dsDNA detected

As expected, lupus nephritis was closely associated with the anti-dsDNA positive group when compared with the anti-dsDNA negative group (OR = 5.98, χ2 = 11.16, P < 0.0008) (Table 1). Lymphopenia, leukopenia, anti-Ro, anti-nRNP and anti-Sm all were associated with the anti-dsDNA group (Table 1). On the other hand, photosensitivity and fibromyalgia were negatively associated with the anti-dsDNA group (Table 1). Other findings that were associated with SLE affecteds from families with anti-dsDNA were higher erythrocyte sedimentation rate (ESR <30 (0–20 NL), 56/71 (79%) vs 20/43 (46%), OR = 4.29, χ2 = 12.62, P < 0.0003) and lower serum complement levels (CH50 < 25 (40–70 NL), 37/71 (52%) vs 8/43 (19%) OR = 4.76, χ2 = 12.59, P < 0.0003). Finally, the nuclear homogeneous ANA pattern (NH) was strongly associated with the anti-dsDNA positive group (47/71 (66%) vs 9/43 (21%), OR = 7.40, χ2 = 21.95, P < 0.00001). Interestingly, none of the randomly selected anti-dsDNA negative patients had positive anti-Sm results. There were only two patients with anti-Sm precipitins in all 43 pedigrees with no SLE affected with anti-dsDNA. Both anti-Sm and anti-nRNP are more frequent in African-American than in European-American SLE patients,17 but in these pedigrees both anti-Sm and anti-nRNP were also increased in the European-American pedigrees (7/37 (19%) vs 0/33 (0%), OR = infinity, χ2 = 6.94, P < 0.008 and 13/37 (35%) vs 2/33 (6%), OR = 8.39, χ2 = 8.76, P < 0.003, respectively).

Screening for linkage

Using a screening set of six inheritance models, we first scanned the genome for linkage in the selected group using the maximum-likelihood model-based method. Seven effects surpassed the recommended threshold for suggestive linkage (LOD 1.9) with two surpassing the threshold for significant linkage (LOD 3.3)18 (Table 2). The genome screening results were dominated by a powerful genetic effect in the 37 European-American pedigrees at 19p13.2 which produced LODmax = 4.93 using a dominantly inherited model with 92% penetrance in women and 49% in men, at a recombination fraction of 0 (θ = 0) and at 100% homogeneity (α = 1.0) (Figure 2). In addition, the 29 African-American pedigrees with an SLE affected that had anti-dsDNA, established linkage with LOD = 3.40 at 18q21.1 (LODmax = 3.40) using a recessively inherited model with 100% penetrance (θ = 0.1, α = 1.0) (Figure 3). There were no linkage effects in these loci for the corresponding anti-dsDNA negative group.

Table 2 Significant (LOD ≥3.3 or P ≤ 0.00002) and suggestive linkages (LOD ≥1.9 or P ≤ 0.0017) identified by genome scan data in SLE pedigrees with at least one affected with a positive anti-dsDNA
Figure 2

Chromosome 19 two-point LOD scores for the 37 European-American pedigrees that contain at least one affected with a positive anti-dsDNA, as detected by Crithidia luciliae kinetoplast imunofluorescence. Best marker: D19s714. LODmax = 4.93 at 36 cM with complete homogeneity (θ = 0). The solid line at LOD = 3.3 is the threshold for significant linkage at the P = 0.05 level in a genome scan.

Figure 3

Chromosome 18 two-point LOD scores for the 29 African-American pedigrees that contain at least one affected with a positive anti-dsDNA, as detected by Crithidia luciliae kinetoplast imunofluorescence. Best marker: D18s858. LODmax = 3.40 at 67 cM (θ = 0.1). The solid line at LOD = 3.3 is the threshold for significant linkage at the P = 0.05 level in a genome scan.

We then performed multipoint linkage analysis using GENEHUNTER-PLUS, which produced results very consistent with the two point linkages and provided further evidence of susceptibility genes residing in these regions (Figures 4 and 5). Among the 37 European-American pedigrees, a LODmax = 3.63 at 36 cM with D19s714 was identified using a dominantly inherited model with 100% and 20% penetrance in females and males, respectively, a disease allele frequency of 5% and 100% homogeneity. When heterogeneity was allowed, HLODmax = 4.0 when 80% of the selected families were linked in this region (Figure 4).

Figure 4

Multipoint parametric LOD scores and the non-parametric linkage (NPL) scores on Chromosome 19 in the 37 European-American pedigrees that contain at least one affected with a positive anti-dsDNA. Best model: dominant with allele frequency of 0.05. Penetrance: female 100% and male 20%. LODmax = 3.63 under homogeneity. HLODmax = 4.0 with α = 80%. NPLmax Score = 1.68 at 26 cM, P-value = 0.05. Peak of LOD and HLOD is at 36 cM; the closest marker is D19S714.

Figure 5

Multipoint parametric LOD scores and the non-parametric linkage (NPL) scores on Chromosome 18 in the 29 African-American pedigrees that contain at least one affected with a positive anti-dsDNA. Best model: recessive with allele frequency of 0.09. Penetrance: 100%. LODmax = −1.73 under homogeneity. HLODmax = 3.68 with α = 60%. NPLmax score = 2.48 at 66 cM, P-value = 0.05. Peak of LOD and HLOD is at 66 cM.

Among the 29 African-American pedigrees, HLODmax = 3.68 at 66 cM on chromosome 18 was identified using a recessive model with penetrance of 100% for both females and males, and a disease allele frequency of 9% when 60% of the families were linked to the locus. Heterogeneity of the linkage prevented the homogeneous model from producing a significant result (LODmax= −1.73) (Figure 5).


Two major susceptibility loci for SLE have been detected by stratifying pedigrees with anti-dsDNA autoantibodies. The more impressive linkage signal, SLED1 (LODmax = 4.93 and HLODmax = 4.0), is at 19p13.2 and operates in the European-American pedigrees. The other convincing linkage signal, SLED2 (HLODmax = 3.68), is at 18q21.1 in the African-American pedigrees. The two-point LOD score results are consistent with multipoint analyses where HLOD = 4.00 at 19p13.2 and HLOD = 3.68 at 18q21.1 for European-American and African-American pedigrees, respectively. How these genes may be related to anti-dsDNA antibodies, however, is not known or evident from these experiments.

Nevertheless, this effort is a successful example of the strategy of stratifying pedigrees by clinical, laboratory and/or demographic features. Here, we used the presence or absence of one of the most specific autoantibodies, ie anti-dsDNA, to isolate genetic effects. The extraordinary clinical and serological heterogeneity of SLE is usually considered to be a disadvantage for genetic studies. Although stratification of pedigrees based on a particular phenotype reduces the number of SLE patients, and consequently must affect the statistical power, these findings demonstrate that stratifying by the presence of anti-dsDNA improves the genetic homogeneity in some situations; thereby more easily reveals genetic effects through improved statistical power.

An association of a positive anti-dsDNA with more severe disease, in particular lupus nephritis, was also strongly supported in this study and may very likely be related to the subsequent linkage effects. Whether this relationship operates through a direct or indirect mechanism is not known.

The association of anti-dsDNA with anti-Sm, anti-nRNP and anti-Ro in these pedigrees is consistent with the possibility that the SLE genetic effects identified (ie SLED1 and SLED2) could trigger a biologic pathway that encourages lupus autoantibody production.

Pathogenic IgG high-avidity anti-dsDNA arising from the partial imitation of foreign antigens, such as infectious agents rather than by DNA from self, has been suggested to be important in the immune mechanisms causing clinical disease in SLE.19 Somatic mutation is thought to drive the immune response from low avidity IgM anti-dsDNA toward high-avidity IgG anti-dsDNA.19 Not only can self-DNA be rendered immunogenic by viral infections, but also by mutations in the enzymes that are responsible for DNA degradation. In fact, deoxyribonuclease 2 (DNase 2) is one of the potential candidate genes located at 19p13.2 partly because mutations in DNase1 have been implicated in lupus by others.20 Indeed, low-activity (DNase2 L) and high-activity (DNase2 H) polymorphisms are known.21 DNase 2 has been implicated in diverse functions including the degradation of foreign DNA, genomic instability, and the mediation of DNA digestion associated with apoptosis.22

DNA methyltransferase 1 is in or near 19p13.2. Environmental agents can also inhibit DNA methylation and predispose to SLE.23 T cells isolated from patients with active lupus have hypomethylated DNA with diminished DNA methyltransferase activity.23 Interaction of the ras-mitogen-activated protein kinase (MAPK) pathway and the levels of this enzyme also may be altered in patients with lupus.24 Moreover, in vitro inhibition of this enzyme can induce an immune complex glomerulonephritis with IgG anti-DNA and antihistone antibodies.25

Calreticulin, a calcium binding multifunctional protein that is a suspected autoantigen that may have a role in transcription regulation26 and in penetration of anti-dsDNA into the cells.27 Heterogeneous nuclear ribonucleoproteins (hnRNPs) and the complement component C3 gene are other attractive candidates in the 19p13.2 region.

Candidate genes for the linkage at 18q21.1 include RAD30, which encodes one of a family of DNA polymerases that plays a pivotal role in the ability of cells to tolerate DNA damage.28 One of the mitogen-activated protein kinase enzymes (MAPK 4) (18q12-21), which their possible interaction pathways with DNA methyltransferase were mentioned above and the anti-apoptotic Bcl-2 gene are located here.

Obviously, there are many genes involved in the pathogenesis of SLE29 and these two linkage effects with SLE are among the enlarging set of genetic effects revealed by pedigree stratification. An effect at 17p13 sufficient to establish linkage (LOD = 3.6) was found in European-American pedigrees stratified by having an SLE affected with vitiligo.30 An effect at 5p15.3 was established (LOD = 5.62) in European-American pedigrees that were also multiplex for reported rheumatoid arthritis (Namjou B et al, unpublished data). Two effects found by stratifying African-American pedigrees multiplex for SLE with thrombocytopenia at 11p13 (Scofield RH et al, unpublished data) and with hemolytic anemia at 11q14 (Kelly JA et al, unpublished data) produce LOD >4.5 and also strongly suggest the action of individual genes in selected subsets of lupus pedigrees. Indeed, other contributions elsewhere in this issue identify other genetic effects, revealed by pedigree stratification.31,32,33

No doubt the clinical, serological, and demographic features of SLE have the potential to reveal many genetic effects. SLE appears to be generated by the concerted action of a relatively large number of genes. Hopefully, the genes responsible for these effects can be identified, thereby providing insight into disease pathogenesis and leading to useful new therapeutic strategies.

Patients and methods


Procedures for the recruitment and enrollment of families in the lupus genetics studies and the genotyping of 307 microsatellite markers have been described previously.6 Criteria for enrollment required that at least two family members meet the 1982 ACR revised criteria for SLE15,16 and that their relationship be potentially informative for linkage with an SLE phenotype. Each potential participant was interviewed and asked to complete an extensive questionnaire. All available medical records were reviewed in the SLE affecteds in order to confirm the diagnosis. In some cases, the participant’s physician was also interviewed. Family members unaffected by SLE completed a less extensive questionnaire in order to be screened for SLE and other autoimmune disease. The ethnicity and percentage of each participant was based on the participants’ self-report.

Association data

A standard set of SLE serologies was collected for each participant in a CLIA approved clinical laboratory at the Oklahoma Medical Research Foundation. Antinuclear antibody titer and pattern were determined on a HEp-2 substrate (Invitrogen) by immunofluorescence following the published procedure.34 The same procedure, except with immunofluorescence of the kinetoplast of C. luciliae, was used to determine anti-dsDNA.35 Precipitating concentrations of antibodies binding to Sm were detected by the classic Oüchterlony immunodiffusion procedure.36 Anti-cardiolipin antibodies were determined by a solid phase assay that is in standard use and follows the usual enzyme-linked immunosorbent procedure.37

Genotyping and error checking

Genomic DNA was isolated from peripheral blood mononuclear cells, buccal cell swabs or lymphoblastoid cell lines using conventional methods after informed consent was obtained. A total of 307 microsatellite markers were genotyped from the Version 8 Weber screening set ( (Amplified fragments were detected using 6% polyacrylamide gels electrophoresed on automated LiCor Model 4000 DNA sequencers. Gel images were collected using Base ImagIR software, version 4.0, and alleles were determined using Gene ImagIR, version 3.52. Genotyping data from 29 pedigrees were obtained by the Mammalian Genotyping Center in Marshfield, WI, USA ( Prior to any linkage analysis, sibling, half-sibling and parent-offspring relationships were confirmed using RELTEST, a feature of the S.A.G.E. 4.0 package, version Beta 3.38,39 This program is based on Markov-process models to calculate pair-specific statistics that estimate average genome-wide allele sharings which classify the relationship for the respected relative pair. For all linkage analyses, the pedigrees were analyzed together and as subsets containing only African-American or European-American pedigrees. For each marker, allele frequencies were estimated by allele counting using the unrelated pedigree members.

Two-point linkage analysis

Two-point LOD scores were calculated using FASTLINK, version 4.1P, and the ANALYZE package.40,41 Individual pedigree-specific LOD scores were generated at different theta values in 0.05 increments from 0.0 (complete linkage) to 0.5 (no linkage). These pedigree LOD scores were then summed together in order to obtain the overall LOD score at each recombination increment (theta). We first applied the microsatellite genotyping data to calculate two-point LOD scores using the classic inheritance-specified model based linkage analysis originally developed by Newton Morton.42 In this approach, six inheritance models were used for screening analysis as described elsewhere:6 recessive at 100% penetrance (R100) and 50% penetrance (R50), dominant at 90% penetrance (D90) and at 50% penetrance (D50%), and dominant and recessive models with mixed penetrances (92% for females and 49% for males) (Rmix and Dmix). The inter marker distances were obtained from the Marshfield marker database ( The SIBPAIR subroutine of the ANALYZE package was implemented to identify any excess allele sharing among affected sibling pairs (affected sibling pairs = 60).

Multipoint linkage analysis

Genetic linkage analysis using GENEHUNTER was used since it allows complete multipoint analysis with a large number of highly polymorphic markers. Both traditional parametric (LOD scores, assuming genetic homo- or heterogeneity) and non-parametric linkage analysis (NPL scores, assessed by comparing the observed set-wise identical-by-descent sharing among all affected family members to that expected under the null hypothesis of no linkage) are available with this approach. In addition, ‘information content mapping’ provides an index of the inheritance information extracted at each point in the genome by the marker genotyped.43 The optimum model was chosen from the set of models constrained by a fixed population prevalence.


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The authors would like to thank the patients and family members who participated in this study and those who referred them to us. Ninety pedigrees (Cohorts A, B and C) were obtained from the Lupus Multiplex Registry and Repository (AR-1-2253) (see

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Correspondence to J B Harley.

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Supported by the NIH (AR12253, AI24717, AR42460, AR45231, AI31584, RR15577) and the US Department of Veterans Affairs.

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Namjou, B., Nath, S., Kilpatrick, J. et al. Genome scan stratified by the presence of anti-double-stranded DNA (dsDNA) autoantibody in pedigrees multiplex for systemic lupus erythematosus (SLE) establishes linkages at 19p13.2 (SLED1) and 18q21.1 (SLED2). Genes Immun 3, S35–S41 (2002) doi:10.1038/sj.gene.6363905

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  • SLE, linkage analysis
  • anti-double stranded DNA
  • Crithidia luciliae

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