Renal disease occurs in 40–75% of systemic lupus erythematosus (SLE) patients and significantly contributes to morbidity and mortality. We used two pedigree stratification strategies to explore the impact of the ACR renal criterion for SLE classification upon genetic linkage with SLE. In both we used SLE as the phenotype. First, we evaluated genome scan data from >300 microsatellite markers in the 75 pedigrees that had at least one SLE affected with the SLE renal criterion. A maximum-likehood parametric model approach produced a maximum screening LOD score of 3.16 at 10q22.3 in the European-American (EA) pedigrees. The African-American (AA) pedigrees obtained a maximum screening LOD score of 2.58 at 11p15.6. A multipoint sib-pair regression analysis produced P = 0.0000008 in EA at 10q22.3 (SLEN1) and P = 0.000001 in AA at 2q34-35 (SLEN2). A second stratification strategy explored the renal criterion in 35 pedigrees with two or more SLE patients with renal disease and produced a LOD score of 3.34 at 11p15.6 in AA (SLEN3). Sib-pair analysis in these 35 pedigrees revealed P = 0.00003 at 4q13.1 in EA, P = 0.00003 at 11p13 and 0.00007 at 3q23 in AA. Thus, multiple genetic linkages are related to the renal criterion in SLE. Of the significant genetic linkages with SLE described herein, those at 10q22.3 in the EA pedigrees (SLEN1) and at 2q34-35 in the AA pedigrees (SLEN2) have not been previously described.
Systemic lupus erythematosus (SLE) is an autoimmune disease with multiple organ involvement and a genetic predisposition. Renal disease occurs in 40–75% of SLE patients within 5 years of disease onset.1 In childhood SLE, nephritis is present in up to 90% of the cases.2 In fact, nephritis is one of the most serious complications of SLE, with a high morbidity and mortality. It has been suggested that the African-American patients have a higher incidence of SLE as well as of SLE nephritis with a less favorable prognosis in comparison to European-Americans.3,4 Patients with focal and diffuse proliferative glomerulonephritis, as described by the World Health Organization classification of SLE nephritis, clearly have an increased risk of developing severe renal impairment.5
Immunologically, 85% of SLE patient sera contain antibodies binding small RNA-proteins including Ro (SS-A) and nRNP.6 Autoantibodies binding nRNP, Ro/SSA and Sm antibodies have been shown to be present at a higher frequency in the African-American SLE patients.7,8 Also, antibodies to double-stranded DNA and low complement are thought to be involved in the pathogenesis of SLE nephritis.9,10,11 Other autoantibodies potentially involved in the pathogenesis of SLE nephritis include those binding the histones, RNA polymerase and C1q.12,13
The evident complexity of the genetics of SLE is a barrier slowing progress. In the hope of simplifying the genetic milieu in SLE, we have stratified pedigrees according to the renal criteria of the SLE affected family members. We analyzed two separate samples of pedigrees for genetic linkage: first, pedigrees containing at least one SLE affected member who also had renal disease from our collection of 160 pedigrees multiplex for SLE, and second, pedigrees that had two or more SLE patients with renal disease.
Seventy-five pedigrees containing at least one SLE patient with renal involvement were ascertained from 160 pedigrees multiplex for SLE to evaluate genetic linkage. Of the 75 pedigrees, there were 102 affected with proteinuria, 62 with cellular casts and 49 SLE patients with proteinuria and cellular casts. When using the microsatellite genotyping data to screen with two-point LOD scores, maximum likelihood model-based linkage analysis identified a screening LOD score of 3.16 at D10s2470 on chromosome 10q22.3 in the European-American (EA) pedigrees (Table 1). This result was produced with a dominant mode of inheritance with 92% female penetrance and 49% male penetrance at no recombination (θ = 0). This LOD score exceeds the accepted threshold for suspected linkage.14 These data were also evaluated using a multipoint sib-pair regression algorithm (SIBPAL2). Particularly significant effects, sufficient to establish linkage, were found at 10q22.3 in the EA with P = 0.0000008 (Table 2, Figure 1) and at 2q34-35 with P = 0.000001 in the African-American (AA) pedigrees (Figure 2).
Ninety-eight EA sib-pairs contributed to the 10q22.3 effect, consisting of 39 concordant affected pairs, 44 discordant pairs and 15 concordant unaffected pairs. Of the 39 concordant affected sibling pairs, 56% shared two alleles, 36% shared one allele and 8% had no allele in common. Among the discordant sibling pairs, 11% shared two alleles, 75% shared one allele and 14% shared zero alleles. Among the concordant unaffected sib-pairs, 47% shared two alleles, 47% shared one and 6% did not share a common allele. Examples of EA pedigrees with their genotyping at D10s2470 (Figure 3) show the range of their contribution toward linkage at this locus.
There were 35 SLE pedigrees with two or more patients with SLE and renal disease. Linkage analysis with SLE as the phenotype (and not SLE nephritis) using the genome scan genotyping data was performed on these pedigrees. The maximum two-point screening LOD score was 3.34 at D11s1984 on chromosome 11p15.6 among the AA pedigrees (Table 3). SIBPAL2 did not establish any linkage using this subset of pedigrees (Table 4). However, in the AA pedigrees, effects were found with P = 0.00003 at D11s1392 on chromosome 11p13 and P = 0.00007 at D3s1744 on chromosome 3q23. In the EA pedigrees, we obtained a P = 0.00003 at D4s3243 on chromosome 4q13.1.
Of the identified genetic linkages related to the renal criterion in SLE, significant effects were found among EA and AA pedigrees separately, and were less impressive when both groups were analyzed together. This emphasizes, again, the racial differences in the genetics of SLE.
Two separate groups of pedigrees were ascertained on the presence of renal disease with the purpose of identifying novel linkages involved in susceptibility to SLE: (1) pedigrees multiplex for SLE that contained at least one SLE affected member who also had renal disease and (2) pedigrees multiplex for SLE with two or more SLE patients with renal disease. Two novel significant linkages (LOD ⩾3.3 or P ⩽0.00002) were identified at 10q22.3 (SLEN1) in the 31 European-American pedigrees and at 2q34-35 (SLEN2) in the 40 African-American pedigrees containing at least one SLE affected member with renal disease. This stratification strategy also identified nine additional suggestive linkages. Analyzing pedigrees with two or more SLE patients with renal disease identified one potentially significant linkage at 11p15.6 (SLEN3) among the African-American pedigrees as well as eight suggestive linkages. The data support the presence of multiple SLE susceptibility loci among pedigrees stratified on the presence of renal disease.
These results suggest either that selecting pedigrees based on clinical criteria will be an invaluable tool for the study SLE genetics or that we have identified false-positive linkages. Two fundamentally different analytical approaches were used to generate these results and three partially independent groups were evaluated (all, EA and AA pedigrees). How much should we inflate the threshold for established linkage to correct for multiple testing? Certainly, SLEN3 at 11p15.6, which is at the threshold, is at greatest risk for being a false-positive linkage. In addition, these are not the only results reported from this collection of pedigrees generated by pedigree stratification. Other linkages of sufficient magnitude to be otherwise established generated by this approach include pedigrees stratified by vitiligo,15 rheumatoid arthritis, anti-double stranded DNA,16 thrombocytopenia and hemolytic anemia (unpublished data). Whether or not the SLEN1, SLEN2 or SLEN3 are present in our data because of a genetic difference or are false-positive linkages will require confirmation by either independent replication in another collection of pedigrees or by finding genetic association in these genomic regions in these (or other) pedigrees distributed in a way that would explain the linkage.
None of these linkages have been previously detected when using SLE as the phenotype without pedigree stratification. A suspected linkage effect at 10p13 has previously been identified17 on the short arm at 10p13 (LOD = 2.24 at D10s197) and was also found in European-Americans, but SLEN1 at 10q22.3 is more than 70 cM away and, therefore, these effects are likely to have different origins.
SLEN2 at 2q34-35 is more than 50 cM centromeric to SLEB2 at 2q37 found by Marta Alarcón-Riquelme and colleagues.18 The distance separating the effects and the different ethnic backgrounds of the pedigrees contributing (Swedish and Icelandic vs African-Americans stratified by nephritis) led us to conclude that the effects are not likely to be from the same genetic origin.
SLEN3 is almost at the p telomere of chromosome 11 (D11s1984 (LOD = 3.35) at 11p15.5). Suggestive linkage has already been identified in this same region (D11s922: LOD = 1.60; D11s4046: LOD = 1.60),17 though D11s1984 was not evaluated in this previous study. In any case, linkage with SLE to 11p15.5 is supported by these results, which reduces the likelihood that SLE3 is a false-positive linkage signal.
Each of these genetic effects, SLEN1, SLEN2 and SLEN3, has some relationship to SLE nephritis. However, the phenotype evaluated was SLE without regard to nephritis. Consequently, we cannot conclude that there is a direct relationship between these genes and nephritis. Of course, the final answer on this issue must await gene identification and directly testing this hypothesis.
For the three genetic effects, SLEN1, SLEN2 and SLEN3, there are a large number of interesting candidate genes. For example, SLEN1 at 10q22.3 is about 7 cM away from Fas (TNFRSF6, APT1, CD95/Apo-1) mutations that are responsible for an autoimmune lymphoproliferative syndrome.19,20 SLEN2 is close to p70, part of the Ku autoantigen, and is reported to be abnormally expressed in SLE.21
We present new evidence for three SLE susceptibility loci: SLEN1 at 10q22.3 in European-American pedigrees, and SLEN2 at 2q34-35 and SLEN3 at 11p15.5 in African-Americans. Each has been detected after stratifying pedigrees multiplex for SLE by the presence of renal disease. The linkages identified herein are susceptibility genes for SLE since they were identified in pedigrees multiplex for SLE and analyzed with SLE as the phenotype. The strategy presented herein demonstrates that pedigree selection based on clinical manifestations strengthen the likelihood of identifying genes and provide important insights into the underlying pathogenesis of complex disease phenotypes.
Patients and methods
Pedigrees were initially ascertained through the Lupus Multiplex Registry and Repository and the other lupus genetics studies underway at the Oklahoma Medical Research Foundation (OMRF) (Cohorts 1, 2, A, B and C). Each pedigree enrolled was required to contain two family members who were related in a way that was informative for genetic linkage and who had SLE. All patients with SLE met at least four of the revised 1982 American College of Rheumatology (ACR) classification criteria for SLE.22 All patient family origins were European-American, African-American or Hispanic. Ethnicity was by self-identification. From this collection of 160 pedigrees, the 75 pedigrees containing at least one SLE affected with nephritis or the 35 pedigrees with two or more SLE affected with nephritis were separately studied for linkage.
Determination of clinical criteria
All available medical records were reviewed for SLE cases. Patients usually completed an extensive questionnaire and were also interviewed by a trained pedigree recruiter, physician’s assistant, or rheumatologist. Clinical data were abstracted using a standardized case review form as previously presented.23
A participant was considered to have renal disease if there was convincing evidence in the medical records review for the presence of proteinuria and/or cellular casts as given in the ACR criterion for SLE nephritis. The resulting sample of pedigrees used for the present study was multiplex for SLE and contained at least one SLE patient with renal disease. This sample consisted of 75 pedigrees (40 African-American (AA), 31 European-American (EA) and four Hispanic (HI)).
A second stratification was performed, and pedigrees with two or more SLE patients who also manifested renal disease were ascertained. Of the original 75 pedigrees, there were 35 pedigrees multiplex for lupus nephritis (20 AA, 12 EA and 3 HI).
DNA isolation and genotyping
Genomic DNA was isolated from peripheral blood mononuclear cells, buccal cell swabs or EBV-transformed cell lines using standard methods. A total of 307 microsatellite markers were typed from the version 8 Weber Screening Set (research.marshfieldclinic.org/genetics/sets/Set8ScreeningFrames.htm) with an average marker spacing of 11 centiMorgans (cM). Polymerase chain reactions were performed as previously described.23
All linkage tests done in this study used SLE as the phenotype. (No linkage analysis was done specifically with SLE nephritis as the phenotype.) Prior to any linkage analysis, pedigree relationships were confirmed by relTEST,24 a feature of the S.A.G.E. 4.0 package, version Beta 3.25
Two-point LOD scores were calculated using FASTLINK, version 4.1P, and ANALYZE.26,27,28 Six different models were used for the screening analysis as previously described.29 Analyses were executed upon selected pedigrees together, as well as separate racial subsets of pedigrees (AA and EA).
Multipoint linkage analysis was performed as previously described23 using the new Haseman-Elston regression method30 implemented in SIBPAL2, a subroutine of S.A.G.E.25 Analyses were executed upon selected pedigrees together, as well as separate racial subsets of pedigrees (AA and EA).
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Special thanks are given to all the families that participated in the study. The study was supported by the National Institutes of Health (AI42460, AR24717, AR45231, AI31584 and AR52221), the Lupus Multiplex Registry and Repository (AR-1-2253) and the US Department of Veterans Affairs.
Grant Support: This work was supported by the National Institutes of Health (AI42460, AR24717, AR45231, AI31584 and AR52221) and the US Department of Veterans Affairs.
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Quintero-Del-Rio, A., Kelly, J., Kilpatrick, J. et al. The genetics of systemic lupus erythematosus stratified by renal disease: linkage at 10q22.3 (SLEN1), 2q34-35 (SLEN2), and 11p15.6 (SLEN3). Genes Immun 3, S57–S62 (2002) doi:10.1038/sj.gene.6363901
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