Fine Mapping of the SLEB2 Locus Involved in Susceptibility to Systemic Lupus Erythematosus
ABSTRACT We have previously reported linkage of systemic lupus erythematosus to chromosome 2q37 in multicase families from Iceland and Sweden. This locus (SLEB2) was identified by linkage to the markers D2S125 and D2S140. In the present study we have analyzed additional microsatellite markers and SNPs covering a region of 30 cM around D2S125 in an extended set of Nordic families (Icelandic, Swedish, and Norwegian). Two-point linkage analysis in these families gave a maximum lod score at the position of markers D2S2585 and D2S2985 (Z = 4.51, PIC = 0.65), by applying a “model-free” pseudo-marker linkage analysis. Based on multipoint linkage analysis in the Nordic families, the most likely location of the SLEB2 locus is estimated to be in the interval between D2S125 and the position of markers D2S2585 and D2S2985, with a peak multipoint lod score of Z = 6.03, assuming a dominant pseudo-marker model. Linkage disequilibrium (LD) analysis was performed using the data from the multicase families and 89 single-case families of Swedish origin, using the same set of markers. The LD analysis showed evidence for association in the single-case and multicase families with locus GAAT3C11 (P < 0.0003), and weak evidence for association was obtained for several markers located telomeric to D2S125 in the multicase families. Thirteen Mexican families were analyzed separately and found not to have linkage to this region. Our results support the presence of the SLEB2 locus at 2q37.
[show abstract] [hide abstract]
ABSTRACT: Systemic lupus erythematosus is the prototype multisystem autoimmune disease. A strong genetic component of susceptibility to the disease is well established. Studies of murine models of systemic lupus erythematosus have shown complex genetic interactions that influence both susceptibility and phenotypic expression. These models strongly suggest that several defects in similar pathways, e.g. clearance of immune complexes and/or apoptotic cell debris, can all result in disease expression. Studies in humans have found linkage to several overlapping regions on chromosome 1q, although the precise susceptibility gene or genes in these regions have yet to be identified. Recent studies of candidate genes, including Fcgamma receptors, IL-6, and tumour necrosis factor-alpha, suggest that in human disease, genetic factors do play a role in disease susceptibility and clinical phenotype. The precise gene or genes involved and the strength of their influence do, however, appear to differ considerably in different populations.Arthritis Research 02/2001; 3(6):331-6.
Fine Mapping of the SLEB2 Locus Involved in Susceptibility
to Systemic Lupus Erythematosus
V. Magnusson,* A.-K. B. Lindqvist,*,1C. Castillejo-Lo ´pez,* H. Kristja ´nsdottir,† K. Steinsson,†
G. Gro ¨ndal,† G. Sturfelt,‡ L. Truedsson,‡ E. Svenungsson,§ I. Lundberg,§ I. Gunnarsson,§
A. I. Bolstad,¶,? H.-J. Haga,¶,** R. Jonsson,¶L. Klareskog,§ J. Alcocer-Varela,††
D. Alarco ´n-Segovia,†† J. D. Terwilliger,‡‡ U. B. Gyllensten,*,§§ and
M. E. Alarco ´n-Riquelme*,§,2
*Department of Genetics and Pathology and Uppsala Genotyping Center, Uppsala University, 751 85 Uppsala, Sweden; †Department
of Rheumatology and Center for Rheumatology Research, Landspitalı ´ nn University Hospital, Reykjavik, Iceland; ‡Department of
Rheumatology and Department of Clinical Microbiology, Lund University Hospital, Lund, Sweden; §Unit for Rheumatology, Karolinska
Hospital, Karolinska, Sweden;
**Section of Rheumatology, Institute of Medicine, Haukeland University Hospital, Bergen, Norway; ††Department of Immunology and
Rheumatology, Instituto Nacional de la Nutricio ´n “Salvador Zubira ´ n,” Mexico City, Mexico; and ‡‡Department
of Psychiatry and Columbia Genome Center, Columbia University, New York, New York
¶Broegelmann Research Laboratory, ?Center for Medical Genetics and Molecular Medicine, and
Received June 13, 2000; accepted September 1, 2000
We have previously reported linkage of systemic lu-
pus erythematosus to chromosome 2q37 in multicase
families from Iceland and Sweden. T his locus (SL E B2)
was identified by linkage to the markers D2S125 and
D2S140. In the present study we have analyzed addi-
tional microsatellite markers and SNPs covering a re-
gion of 30 cM around D2S125 in an extended set of
Nordic families (Icelandic, Swedish, and Norwegian).
T wo-point linkage analysis in these families gave a
maximum lod score at the position of markers D2S2585
and D2S2985 (Z ? 4.51, PIC ? 0.65), by applying a
“model-free” pseudo-marker linkage analysis. Based
on multipoint linkage analysis in the Nordic families,
the most likely location of the SL E B2 locus is esti-
mated to be in the interval between D2S125 and the
position of markers D2S2585 and D2S2985, with a peak
multipoint lod score of Z ? 6.03, assuming a dominant
pseudo-marker model. L inkage disequilibrium (L D)
analysis was performed using the data from the mul-
ticase families and 89 single-case families of Swedish
origin, using the same set of markers. T he L D analysis
showed evidence for association in the single-case and
multicase families with locus GAAT 3C11 (P < 0.0003),
and weak evidence for association was obtained for
several markers located telomeric to D2S125 in the
multicase families. T hirteen Mexican families were
analyzed separately and found not to have linkage to
this region. Our results support the presence of the
SL E B2 locus at 2q37.
© 2000 Academic Press
Systemic lupus erythematosus (SLE) is an autoim-
mune inflammatory disease potentially involving mul-
tiple organ systems such as skin, joints, kidney, and
brain. Characteristic of the disease is the expression of
a variety of autoantibodies, in particular those against
double-stranded DNA. The prevalence of SLE is ap-
proximately 65/100,000 in Sweden and Iceland (Ståhl-
Hallengren et al., 2000; Gudmundsson and Steinsson,
1990; J onsson et al., 1990) but is potentially higher in
African-Americans (Hochberg, 1985). SLE
prevalent among women, with a female-to-maleratioof
8–9:1. Theageof onset appears tobehigher in patients
of Scandinavian origin (Ståhl-Hallengren et al., 2000)
with a median age at onset of 40 years compared to
patients from other populations (Hochberg, 1985), with
a peak incidence between 35 and 74 years of age. The
etiology of the disease is unknown (Alarco ´n-Segovia
and Alarco ´n-Riquelme, 1998), but epidemiological
studies clearly suggest genetic influence in the suscep-
tibility to SLE. The concordance rate in monozygotic
twins is estimated to be 25–69%, which is at least
10-fold higher than in dizygotic twins (Block, 1993;
Deapen et al., 1992). However, interaction of genetic
and environmental factors has been suggested (Alar-
The URLs for data in this article are as follows: International RH
MappingConsortium at NCBI,
genemap/; The Human Genome Mapping Resource Center, http://
www.hgmp.ac.uk; theGeneticLocation Database(LDB), http://cedar.
genetics.soton.ac.uk; and The Cooperative Human Linkage Center
1Present address: Department of Cell and Molecular Biology, Sec-
tion for Medical Inflammation Research, Lund University, Sweden.
2Towhom correspondence should be addressed at the Department
of Genetics & Pathology, Unit of Medical Genetics, Rudbeck Labo-
ratories, Dag Hammarskjo ¨lds va ¨g 20, 751 85, Uppsala, Sweden.
Telephone: ?46 18 4714805. Fax: ?46 18 4714808. E-mail:
Genomics 70, 307–314 (2000)
doi:10.1006/geno.2000.6374, available online at http://www.idealibrary.com on
Copyright © 2000 by Academic Press
All rights of reproduction in any form reserved.
co ´n-Segovia and Alarco ´n-Riquelme, 1998; J ames et al.,
Efforts to understand the genetics of SLE have been
attempted through association studies of various can-
didate genes, among them the MHC class II and class
III genes, as well as various genes encoding molecules
with possibly relevant immunological functions, such
as the Fc? receptors IIA and IIIA, IL-10, IL-6, T-cell
receptors ? and ?, and mannose-binding protein
(Lindqvist and Alarco ´n-Riquelme, 1999; Tan and Ar-
nett, 1998). The HLA DR and/or DQ genes seem to
more strongly influence the production of specific auto-
antibodies than the overall SLE
(Lindqvist and Alarco ´n-Riquelme, 1999; Tan and Ar-
nett, 1998; Arnett and Reveille, 1992). Deficiency of the
MHC class III complement genes C2 and C4 and non-
MHC C1q has been associated with the disease in
several populations. In particular, partial deficiency of
C4A is frequently found in SLE patients and seems to
be a genetic risk factor for the disease (Arnett and
Searches for putative susceptibility loci by genome-
wide linkage analysis have been performed in several
mouse models (Drake et al., 1994; Hirose et al., 1994;
Morel et al., 1994; Kono et al., 1994; Watson et al.,
1992) as well as in families with SLE (Gaffney et al.,
1998; Moser et al., 1998; Shai et al., 1999; Lindqvist et
al., 2000). Theresults obtained for both themurineand
the human diseases suggest the presence of multiple
susceptibility loci and extensive genetic heterogeneity
(Lindqvist and Alarco ´n-Riquelme, 1999). The genome
scans have not yet resulted in the identification of
susceptibility genes, but studies of congenic strains
with mouse lupus have provided insight into the puta-
tive effects of certain loci (Morel et al., 1996; Wakeland
et al., 1997).
We have previously reported a genome-wide screen
in which we identified a chromosomal location with a
putative susceptibility locus for SLE in Swedish and
Icelandic families (Lindqvist et al., 2000) located in the
2q37 region. We obtained maximum two-point lod
scores of Z ? 4.24 and Z ? 3.53 for markers D2S125
and D2S140, respectively. This locus has been named
SLEB2. We have now analyzed additional markers
covering a distance of approximately 30 cM surround-
ing D2S125, to refine the estimate of the location of
SLEB2 through the use of two-point and multipoint
linkage and linkage disequilibrium analysis. We have
extended our family material of Nordic origin toa total
of 30 multicase families and 89 single-case families. To
test the effects of this locus in a very different genetic
population, we also analyzed 13 Mexican multicase
families. Our results support the presence of a suscep-
tibility locus residing in the 2q37 region in families of
Nordic origin and underscore the importance of using
family cohorts from genetically related populations to
localize genes for complex diseases.
MATERIALS AND METHODS
present study is described in Table 1. Multicase families were se-
lected as those families having at least two or more individuals
fulfilling four or more of the 1982 ACR criteria for SLE (Tan et al.,
1982). The majority of families from Iceland and Sweden has been
described previously (Lindqvist et al., 2000). Pedigree information
from the Icelandic genealogy database made it possible to connect
The complete family material used in the
T ABL E 1
Composition of the F amily Material
PopulationNo. of familiesNo. of SLE patients
PopulationNo. of families Parents available
T ABL E 2
General Description of the SNPs
Locus AccessionGeneNucleotide pos.Nucleotide changeAllele frequencies (%/%)
RNA for type VI collagen ? chain
Homo sapiens mRNA for YSK1
mRNA for KIAA0943 protein
Human thymidylate kinase
Human thymidilate kinase
MAGNUSSON ET AL.
three additional Icelandic individuals affected with SLE in 2 of the
families that we have already studied (Lindqvist et al., 2000). In
addition, 3 new multicase families have been identified and added to
the present study, which represents nearly all expected SLE multi-
case families in Iceland (between 5 and 15% of all SLE patients have
a first- or second-degree relative with SLE; Arnett and Reveille,
1992). Four more families were also added to the cohort of Swedish
families described before (Lindqvist et al., 2000). All Swedish fami-
lies, including the 89 single-case families, originate from Southern or
Central Sweden. Furthermore, the grandparents of all patients in
the single-case families were also born in Sweden (to eliminate
heterogeneity due to possible inclusion of recent immigrants). Six
Norwegian families were also included, originating in various parts
of the country, but with all grandparents born in Norway. Thirteen
multicase families from the Mexico City area were analyzed sepa-
rately. All such families have grandparents born in Mexico and are
mostly Mexicans of Spanish-Amerindian admixture. All individuals,
patients and their relatives, have given informed consent for this
Genotyping of microsatellites.
D2S125 are included in the Weber set 6 and were previously used in
the genome scan (Lindqvist et al., 1996, 2000). GATA178G09 and
GAAT3C11 are from the Cooperative Human Linkage Center (Shef-
field et al., 1995). D2S345, D2S2285, D2S2253, D2S1397, D2S140,
and D2S2338 are from Ge ´ne ´thon (Dib et al., 1996; Gyapay et al.,
1996). COL6A3 was recently described (Pan et al., 1998), as were
D2S2585, D2S2985, and D2S2986, which arelocated at thetelomeric
region of 2q (Rosenberg et al., 1997). Primers were synthesized by
Interactiva Biotechnology (Ulm, Germany) with a conjugated amid-
ite fluorophore at the 5? end of the downstream primer. Each PCR
was optimized for use on 877 ABI instruments (Applied Biosystems,
Inc., Foster City, CA). Fragment lengths were defined using an ABI
377 sequencer with GeneScan program, version 2.0 (Applied Biosys-
tems, Inc.). Allele-calling was performed using the Genotyper pro-
gram, version 2.0 (Applied Biosystems, Inc.), and Mendelian segre-
gation of marker alleles was verified using the GAS program (Alan
Young, Oxford University).
Markers D2S1363, D2S427, and
Search for single-nucleotidepolymorphisms within 2q37.
GenMap98 from theInternational RH Mapping Consortium at NCBI
was used tocheck every expressed sequence tag (EST) or gene found
within the 2q37 cytogenetic region, between D2S345 (AFM288vb1)
and stsSG29476. This region covers 19 cM (sex-averaged). EST se-
quences were retrieved and aligned using ESTBlast software (Gill et
al., 1997), which is available through registration at the UK Human
Genome Mapping Project Resource Center (Cambridge, UK). The
alignments obtained were visually inspected for the presence of
potential polymorphisms. Mismatched bases were visualized when
possible in the sequence chromatogram using the ESTace viewer
from the Genome Sequencing Center, Washington University School
of Medicine (St. Louis, MO). Biallelic markers were chosen based on
two criteria: (a) that more than seven large (?200 bp) EST entries
could be aligned with the original gene/EST sequence with more
than 85% homology and (b) that at least two entries contained the
less frequent allele.
Based on abovecriteria, weidentified 36 potential SNP markers. A
dynamic allele-specific hybridization (DASH) assay (see below) was
designed for each of these SNPs and tested in a panel of 10 Nordic
unrelated individuals. For 6 of the putative SNPs, differences could
not be unambiguously ascertained between the match and the mis-
match probes with the DASH assay, and in 16 of them, no variation
was detected. Fourteen SNPs were further analyzed in 50 unrelated
individuals of Swedish origin and their allelic frequencies are shown
T ABL E 3
Sequences of the Amplification Primers and DASH Match and Mismatch Probes
LocusPrimer 1Primer 2 Probe 1 Probe 2
T ABL E 4
Microsatellite Marker Order and Intermarker
Distances According to the Summary Map from L DB
gaat3cll (D2S2949) 0.57
Note. nd, no data available.
FINE MAPPING OF A LOCUS FOR SLE IN 2q37
in Table 2. The primers and probes used for the SNP genotyping
assays are shown in Table 3.
Genotyping of the SNPs.
DASH method with minor modifications (Howell et al., 1999). The
PCRs were performed in a 20-?l reaction including 35 ng of genomic
DNA, 9.6 pmol of unlabeled upstream primer, and 2.4 pmol of biotin-
labeled downstream primer. The PCR cycling conditions were as
follows: 95°C for 8 min and then 2 cycles of 95°C for 20 s, 60°C for 35 s
followed by 2 cycles of 95°C for 20 s, 59°C for 35 s and finally 40 cycles
of 95°C for 20 s, 58°C for 35 s with a final extension of 10 min at 68°C.
For the SNP at AA15760 the annealing temperature was reduced
3°C, ending at 55°C. After amplification, a volumeof 10 ?l of thePCR
was bound overnight to streptavidin-coated microtiter plates (Hy-
baid Limited, Middlesex, UK). After the nonbiotinylated strand was
washed off with alkali, hybridization was performed separately for
each of the allele-specific probes (match and mismatch probes), at
low temperature. The sample was then steadily heated while fluo-
rescence was monitored using a Perkin–Elmer 7700 TaqMan instru-
ment (Applied Biosystems, Inc.). The amount of fluorescence is pro-
portional tothe amount of hybridized double-stranded DNA, and the
difference in hybridization between the matched and the mis-
matched probes can be analyzed.
The SNPs were genotyped using the
ing the MLINK (Morton, 1955; Ott, 1991) program (FASTLINK 4.0;
Cottingham et al., 1993; Scaffer et al., 1994) and the ANALYZE
program package (Terwilliger, 1995, 2000; Go ¨ring and Terwilliger,
2000a). The presence of linkage heterogeneity was tested for using
theadmixturetest (Smith, 1961). Multipoint analysis was performed
using LINKMAP (FASTLINK 4.0).
Since the evidence for linkage to the region was previously de-
tected under the assumption of dominant inheritance (Lindqvist et
al., 2000), the two-point as well as the multipoint linkage analysis
was performed assuming a dominant mode of inheritance as well.
Because it is impossible toknow the mode-of-inheritance parameters
of a complex disease, and because we assume that the phenotype
“affected” contributed significantly more predictive value (vis-a `-vis
the underlying disease locus genotypes) than the phenotype “unaf-
fected,” we used an affected-only dominant pseudo-marker analysis,
as described elsewhere (Go ¨ring and Terwilliger, 2000a). Such anal-
ysis is shown tobe equivalent totraditional “model-free” approaches
on simple pedigree structures and to be more powerful in large
pedigrees, such as the ones we have in this study (Lindqvist et al.,
2000). Allele frequencies were estimated by allele counting in the
pedigree material (Smith, 1957; Go ¨ring and Terwilliger, 2000b). The
marker order and intermarker distances were obtained from the
summary map of the genetic location database (LDB), with the
exception of the distance between D2S125 and D2S140, which is
assumed tobe 3 cM (telomeric) (Nancy J . Cox, University of Chicago,
pers. comm., J une 1999). The relative positions of markers from
D2S2285 to the SNP T79464b were based on a physical map that
was kindly provided by Dr. Graeme Bell, University of Chicago
(Horikawa et al., 2000).
Two-point linkage analysis was performed us-
Linkage disequilibrium analysis.
equilibrium was tested using a haplotype relative risk study design
(Falk and Rubinstein, 1987; Terwilliger and Ott, 1992). The marker
alleles transmitted from both heterozygous and homozygous parents
toaffected offspring were used as the case sample and the nontrans-
mitted alleles were treated as an independent genetically matched
control sample. The likelihood was then computed following the
method of Terwilliger (1995) as a function of ?, the proportion of
association observed in the sample. The haplotype relative risk test
was applied as it is somewhat more powerful than the standard TDT
method (Terwilliger, 1995).
The presence of linkage dis-
We have previously reported evidence of linkage toa
region on chromosome 2q37 with markers D2S125 and
D2S140 in multicase SLE families from Iceland and
Sweden. In the present study we have typed additional
markers in the region and included new Nordic fami-
lies (total n ? 30). The heterozygosity and microsatel-
lite marker order we assumed were taken from the
summary map of the genetic LDB as shown in Table 4.
Two-point lod scores for 15 microsatellitemarkers on
chromosome 2q37 are shown in Table 5. The maximum
two-point lod score was obtained with marker D2S125
(Z ? 3.59), followed by marker D2S140 (Z ? 2.72) using
a dominant pseudo-marker analysis (Go ¨ring, 2000a).
Markers D2S2585 and D2S2985 are located at the
same position according to all available genetic maps.
For this reason they were tested jointly, assuming a
recombination fraction of ? ? 0 between them. The
two-point lod score obtained for the joint marker
D2S2585/D2S2985 was Z ? 4.51. Multipoint linkage
analysis indicated the best estimate of the position of
thelocus tobeapproximately 4 cM telomeric toD2S125
with a peak lod score of Z ? 6.03 close tothe combined
marker D2S2585/D2S2985 using the same inheritance
model (Fig. 1).
To analyze the region further by linkage disequilib-
rium (LD) mapping, we used a likelihood based HRR
T ABL E 5
T wo-Point L inkage Analysis R esults for the Nordic
Multicase F amilies
Note. Maximum lod scores obtained by two-point linkage analysis
using MLINK (FASTLINK 4.0) as implemented by the ANALYZE
package applying dominant inheritance, affected-only analysis, and
disease gene frequency of PD ? 0.002. LodHom, two-point linkage
analysis assuming homogeneity; LodHet, two-point linkage analysis
allowing for heterogeneity. Allele frequencies for each marker were
obtained by allelecounting in pedigreematerial. ThePIC valueis the
information content of each marker for the multicase family mate-
aD2S2585/D2S2985 were treated as a single marker. The PIC
value for these two markers combined was estimated for the haplo-
types, which may be a slight underestimate of the real PIC value
when they are analyzed jointly in a two-point analysis with ? ? 0
MAGNUSSON ET AL.
ratio test (Terwilliger, 1995). LD mapping was per-
formed using the microsatellite markers and SNPs. A
group of 89 single-case families was also included.
Fourteen SNPs located in genes and ESTs surrounding
D2S125 were analyzed. A physical map made available
to us (G. Bell, Horikawa et al., 2000) allowed us to
infer the most likely order of the following markers:
D2S2285, D2S2253, D2S125, D2S140, stsAA009670,
Cda0fd11, StsG314, and T79464 (a and b). These loci
aremarked with an asterisk in Table6. Themost likely
physical order of the remaining loci was inferred from
LDB and GeneMap98 (NCBI).
The multicase and single-case families were first
analyzed separately and then jointly. In the multicase
families, the strongest evidence for association was
found with microsatellite marker D2S2585 (P ? 0.003,
HRR-LTR), but weak evidence was seen also with
D2S2986 (P ? 0.01, HRR-2xn) and the SNP W52438b
and Cda0fd11 (P ? 0.02 and P ? 0.01, respectively
with HRR-2xn). A tendency toward lower P values was
observed for the region telomeric from D2S125, al-
though none reached statistical significance after cor-
rection for multiple testing using Bonferroni. In the
single-case families, the microsatellite GAAT3C11
showed strongest association (P ? 0.002) that persisted
when multicase and single-case families were com-
bined (P ? 0.0003) (Table 6). None of these findings,
however, remained statistically significant after cor-
rection for multiple testing.
To test for linkage to the 2q37 locus in families from
a different population, we genotyped a set of families
from the Mexican population (n ? 13). The maximum
two-point lod score obtained was for D2S2338 (Z ?
0.71) (Table 7), while no other marker showed any
indication of linkage.
Herein we have sought tostrengthen the linkage of
the SLE B2 locus on chromosome 2q37, described
initially in a genome scan performed on Swedish and
Icelandic families (Lindqvist et al., 2000). We have
now analyzed additional microsatellite markers, 5 of
which were included in our genome scan report, and
14 SNPs covering a distance of approximately 30 cM
in a total of 30 multicase families of Nordic origin.
The two-point and multipoint analyses using the
microsatellite markers suggest an approximate loca-
tion of the SLE B2 locus near D2S125. The multicase
families and a new group of single-case families were
analyzed for LD with microsatellite markers and
SNPs. The strongest evidence of LD was found in the
multicase families with D2S2585 (P
GAAT3C11 gave the strongest evidence for associa-
tion in single-case families and when all families
were combined. The exact location of GAAT3C11,
however, is unknown, but according to the available
genetic maps, this marker is centromeric to D2S125.
We are at present trying to define its exact location
through physical mapping. Although none of the P
values remained significant after correction for mul-
F IG. 1.
number of alleles for each marker was reduced to facilitate the calculations. Reduced allele frequencies were used in the calculation. The
marker order and theintermarker distances wereobtained from theLDB summary map. On all genetic maps availablethemarkers D2S2585
and D2S2985 were located at the same position, so the multipoint linkage analysis was performed using the markers jointly. The lod-3
support interval is indicated.
Multipoint curve. Multipoint analysis assuming dominant inheritance (PD ? 0.002) using LINKMAP (FASTLINK 4.0). The
FINE MAPPING OF A LOCUS FOR SLE IN 2q37
tiple tests, several markers telomeric to D2S125
showed a tendency for association. Although at the
present moment we cannot delimit the region with
confidence, we have confirmed the presence of this
susceptibility locus for SLE in our group of 30 Nordic
families. Independent confirmation of suggestive
linkage to D2S125 in American multicase families
for SLE has been recently obtained (J . B. Harley,
comm., May 2000).
The use of SNPs for fine mapping of susceptibility
loci for complex diseases has been widely proposed
particularly because SNPs occur at a higher fre-
quency than microsatellites. However, the utility of
SNPs in fine mapping depends on several factors
such as the degree of linkage disequilibrium between
the SNP and the actual disease mutation and the
strength of the effect of the actual mutation in the
disease. The SNPs we have studied here are located
within genes and transcript covering 20 cM sur-
rounding D2S125, but may be too far away from the
disease mutation. A recent study analyzing SNPs
surrounding the APOE allele involved in Alzheimer
disease could detect association with SNPs only if as
close as 50 kb from the disease allele with a weak
association showing at 400 kb from APOE (Martin et
al., 2000). We are at present completing the physical
map for 2q37.3 and searching for new SNPs in the
The difference between the linkage results for fam-
ilies of Nordic and those of Mexican descent might
lead one to believe that this locus confers suscepti-
bility for development of SLE mainly in Nordic pop-
ulations. Presumably, the common genetic back-
ground of the Nordic families made it possible to
detect the effect of this locus, which might otherwise
not have been possible using a more admixed popu-
lation with a greater degree of genetic heterogeneity.
A much larger number of Mexican families is needed
to demonstrate the presence of this locus in this
population. As is evident from the mouse lupus mod-
els as well as the recent results from genome-wide
screens of human SLE , the disease exhibits pro-
nounced genetic heterogeneity.
rather likely that susceptibility loci may differ in
nature between ethnic populations.
T ABL E 6
L inkage Disequilibrium Analysis in the Nordic Multicase and Single-Case F amilies
MulticaseSingle caseMulticase and single case
HRR-LRTHRR-2xnHRR-LTR HRR-2xnHRR-LTR HRR-2xn
Note. Likelihood-based haplotype relative risk test for association as performed by the HRRLAMP program applied by the ANALYZE
package (J . Terwilliger). The likelihood ratio test, HRR-LRT, and the 2xn table ?2test; HRR-2xn results presented as P values. Significant
values are in bold (P ? 0.05). The SNP N31909 is not included due togenotyping problems. The positions of the loci marked with an asterisk
have been confirmed by physical mapping (G. I. Bell, University of Chicago, pers. comm., Sept 2000; Horikawa et al., 2000).
aMarker GAAT3C11 is located centrometric to D2S2253 according to GeneMap98.
MAGNUSSON ET AL.
This work has been supported by European Commission Grant
BMH4-98-3489 and NorFa Grant 00.15.062-O, as well as by the
following individual grants: the Swedish Medical Research Council
toM.E.A.-R. (Grant 12673) and G.S. and L.T. (Grant 13489); the Åke
Wibergs Stiftelse toM.E.A.-R.; the King Gustaf V:80th J ubilee Fond
to M.E.A.-R.; the Margareta Rheumatology Research Foundation to
I.L.; the Swedish National Association against Rheumatism to
M.E.A.-R., G.S., L.T., and I.L.; the Landspitalı ´ nn and University of
Iceland Research Funds to K.S.; the Research Council of Norway to
R.J . (Grant 115563/320); and the Hitchings–Elion fellowship from
the Burroughs Wellcome Fund toJ .D.T. The authors are indebted to
Dr. GraemeBell and YukioHorikawa from theUniversity of Chicago
for kindly providing physical mapping information on 2q37 prior to
Alarco ´n-Segovia, D., and Alarco ´n-Riquelme, M. E. (1998). Ethio-
pathogenesis of systemic lupus erythematosus: A tale of three
troikas. In “Systemic Lupus Erythematosus” (R. G. Lahita, Ed.),
pp. 55–69, Academic Press, San Diego.
Arnett, F. C., and Reveille, J . D. (1992). Genetics of systemic lupus
erythematosus. Rheum. Dis. Clin. North Am. 18: 865–892.
Block, S. R. (1993). Twin studies: Genetic factors are important.
Arthritis Rheum. 36: 135–136.
Cottingham, R. W., J r., Idury, R. M., and Schaffer, A. A. (1993). Fast
sequential genetic linkage computations. Am. J . Hum. Genet. 53:
Deapen, D., Escalante, A., Weinrib, L., Horwitz, D., Bachman, B.,
Roy-Burman, P., et al. (1992). A revised estimate of twin concor-
dance in systemic lupus erythematosus. Arthritis Rheum. 35: 311–
Dib, C., Faure, S., Fizames, C., Samson, D., Drouot, N., Vignal, A., et
al. (1996). A comprehensive genetic map of the human genome
based on 5,264 microsatellites. Nature 380: 152–154.
Drake, C. G., Babcock, S. K., Palmer, E., and Kotzin, B. L. (1994).
Genetic analysis of the NZB contribution to lupus-like autoim-
mune disease in (NZB X NZW)F1 mice. Proc. Natl. Acad. Sci. USA
Falk, C. T., and Rubinstein, P. (1987). Haplotype relative risk: An
easy reliable way to construct a proper control sample for risk
calculations. Ann. Hum. Genet. 51: 227–233.
Gaffney, P. M., Kearns, G. M., Shark, K. B., Ortmann, W. A., Selby,
S. A., Malmgren, M. L., et al. (1998). A genome-wide search for
susceptibility genes in human systemic lupus erythematosus sib-
pair families. Proc. Natl. Acad. USA 95: 14875–14879.
Gaffney, P. M., Ortmann, W. A., Selby, S. A., Shark, K. B., Ockenden,
T. C., Rohlf, K. E., et al. (2000). Genome screening in human
systemic lupus erythematosus: Results from a second Minnesota
cohort and combined analysis of 187 sib-pair families. Am. J . Hum.
Genet. 66: 547–556.
Gill, R. W., Hodgman, T. C., Littler, C. B., Oxer, M. D., Montgomery,
D. S., Taylor, S., and Sanseau, P. (1997). A new dynamic tool to
perform assembly of expressed sequence tags (ESTs). Comput.
Appl. Biosci. 13: 453–457.
Go ¨ring, H. H. H., and Terwilliger, J . D. (2000a). Linkage analysis in
the presence of errors. IV. J oint pseudomarker analysis of linkage
and/or linkage disequilibrium on a mixture of pedigrees and sin-
gletons when the mode of inheritance cannot be accurately speci-
fied. Am. J . Hum. Genet. 66: 1310–1327.
Go ¨ring, H. H. H., and Terwilliger, J . D. (2000b). Linkage analysis in
the presence of errors. III. Marker loci and their map as nuisance
parameters. Am. J . Hum. Genet. 66: 1298–1309.
Go ¨ring, H. H. H., and Terwilliger, J . D. (2000c). Linkage analysis in
the presence of errors. II. Marker locus genotyping errors modeled
with hypercomplex recombination fractions. Am. J . Hum. Genet.
Gudmundsson, S., and Steinsson, K. (1990). Systemic lupus ery-
thematosus in Iceland 1975 through 1984. A nationwide epidemi-
ological study in an unselected population. J . Rheumatol. 17:
Gyapay, G., Schmitt, K., Fizames, C., J ones, H., Vega-Czarny, N.,
Spillet, D., et al. (1996). A radiation hybrid map of the human
genome. Hum. Mol. Genet. 5: 339–346.
Hirose, S., Tsurui, H., Nishimura, H., J iang, Y., and Shirai, T.
(1994). Mapping of a gene for hypergammaglobulinemia to the
distal region on chromosome 4 in NZB mice and its contribution to
systemic lupus erythematosus in (NZBxNZW)F1 mice. Int. Immu-
nol. 6: 1857–1864.
Hochberg, M. (1985). The incidence of systemic lupus erythematosus
in Baltimore, Maryland, 1970–1977. Arthritis Rheum. 28: 80–86.
Horikawa, Y., Oda, N., Cox, N. J ., Li, X., Orho-Melander, M., Hara,
M., et al. (2000). Genetic variation in thegeneencoding Calpain-10
is associated with type 2 diabetes mellitus. Nat. Genet. 26: 163–
Howell, W. M., J obs, M., Gyllensten, U., and Brookes, A. J . (1999).
Dynamic allele-specific hybridization. Nat. Biotechnol. 17: 87–88.
J ames, J . A., Kaufman, K. M., Farris, A. D., Taylor-Albert, E.,
Lehman, T. J ., and Harley, J . B. (1997). An increased prevalenceof
Epstein–Barr virus infection in young patients suggests a possible
etiology for systemic lupus erythematosus. J . Clin. Invest. 100:
J onsson, H., Nived, O., Sturfelt, G., and Silman, A. (1990). Estimat-
ing the incidence of systemic lupus erythematosus in a defined
population using multiple sources of retrieval. Br. J . Rheumatol.
Kainulainen, K., Perola, M., Terwilliger, J . D., Kaprio, J ., Kosken-
vuo, M., Syva ¨nen, A. C., et al. (1999). Hypertension 33: 844–849.
Kono, D. H., Burlingame, R. W., Owens, D. G., Kuramochi, A.,
Balderas, R. S., Balomenos, D., et al. (1994). Lupus susceptibility
loci in New Zealand mice. Proc. Natl. Acad. Sci. USA 21: 10168–
Lindqvist, A. K., Magnusson, P. K., Balciuniene, J ., Wadelius, C.,
Lindholm, E., Alarco ´n-Riquelme, M. E., et al. (1996). Chromosome-
T ABL E 7
T wo-Point L inkage Analysis in the Mexican Cohort
Note. Two-point maximum lod scores obtained by MLINK
(FASTLINK 4.0) computed using the ANALYSIS package under
dominant inheritance (P ? 0.002) and affected-only analysis. Lod-
Hom, assuming homogeneity; LodHet, allowing for heterogeneity.
Allele frequencies for each marker were obtained by allele counting
in the pedigree material itself.
FINE MAPPING OF A LOCUS FOR SLE IN 2q37
specific panels of tri- and tetranucleotide microsatellite markers
for multiplex fluorescent detection and automated genotyping:
Evaluation of their utility in pathology and forensics. GenomeRes.
Lindqvist, A. K., and Alarco ´n-Riquelme, M. E. (1999). The genetics of
systemic lupus erythematosus. Scand. J . Immunol. 50: 562–571.
Lindqvist, A. K. B., Steinsson, K., J ohanneson, B., Kristjansdottir,
H., Gro ¨ndal, G., Svenungsson, E., Lundberg, I., et al. (2000). A
susceptibility locus for systemic lupus erythematosus (hSLE1) in
chromosome 2q. J . Autoimmun. 14: 169–178.
Martin, E. R., Gilbert, J . R., Lai, E. H., Riley, J ., Rogala, A. R.,
Slotterbeck, B. D., et al. (2000). Analysis of association at a single
nucleotidepolymorphisms in theAPOE region. Genomics 63: 7–12.
Morel, L., Rudofsky, U. H., Longmate, J . A., Schiffenbauer, J ., and
Wakeland, E. K. (1994). Polygenic control of susceptibility to mu-
rine systemic lupus erythematosus. Immunity 1: 219–229.
Morel, L., Yu, Y., Blenman, K. R., Caldwell, R. A., and Wakeland,
E. K. (1996). Production of congenic mouse strains carrying
genomic intervals containing SLE-susceptibility genes derived
from the SLE-prone NZM2410 strain. Mamm. Genome7: 335–339.
Morton, N. E. (1955). Sequential tests for detection of linkage. Am. J .
Hum. Genet. 7: 277–318.
Moser, K. L., Neas, B. R., Salmon, J . E., Yu, H., Gray-McGuire, C.,
Asundi, N., et al. (1998). Genome scan of human systemic lupus
erythematosus: Evidencefor linkageon chromosome1qin African-
American pedigrees. Proc. Natl. Acad. Sci. USA 95: 14869–14874.
Ott, J . (1991). “Analysis of Human Linkage,” J ohns Hopkins Univ.
Press, New York.
Pan, T. C., Zhang, R. Z., Speer, M. C., and Chu, M. L. (1998). CA
repeat polymorphism of the COL6A3 gene on chromosome 2q37.
Hum. Hered. 48: 235–236.
Risch, N. (1987). Assessing the role of HLA-linked and unlinked
determinants of disease. Am. J . Hum. Genet. 40: 1–14.
Rosenberg, M., Hui, L., Ma, J ., Nusbaum, H. C., Clark, K., Robinson,
L., et al. (1997). Characterization of short tandem repeats from
thirty-one human telomeres. Genome Res. 7: 917–923.
Scaffer, A. A., Gupta, S. K., Shiram, K., and Cottingham, R. W., J r.
(1994). Avoiding recomputation in genetic linkage analysis. Hum.
Hered. 44: 225–237.
Shai, R., Quismorio, F. P., J r., Li, L., Kwon, O. J ., Morrison, J .,
Wallace, D. J ., et al. (1999). Genome-wide screen for systemic
lupus erythematosus susceptibility genes in multiplex families.
Hum. Mol. Genet. 8: 639–644.
Sheffield, D. A., Weber, J . L., Beutow, K. H., Murray, J . C., Even,
D. A., Wiles, K., et al. (1995). A collection of tri- and tetranucleotide
repeat markers used to generate high quality, high resolution
human genome wide linkage maps. Hum. Mol. Genet. 4: 1837–
Smith, C. A. B. (1957). Counting methods in genetical statistics. Ann.
Hum. Genet. 21: 254–276.
Smith, C. A. B. (1961). Homogeneity test for linkage data. Proc.
Second Int. Congr. Hum. Genet. 1: 212–213.
Ståhl-Hallengren, C., J o ¨nsen, A., Nived, O., and Sturfelt, G. (2000).
Incidence studies of systemic lupus erythematosus in Southern
Sweden: Increasing age, decreasing frequency of renal manifesta-
tions and good prognosis. J . Rheumatol. 27: 685–691.
Tan, E. M., Cohen, A. S., Fries, J . F., Masi, A. T., McShane, D. J .,
Rothfield, N. F., et al. (1982). The 1982 revised criteria for the
classification of systemic lupus erythematosus. Arthritis Rheum.
Tan, F. K., and Arnett, F. C. (1998). The genetics of lupus. Curr.
Opin. Rheumatol. 10: 399–408.
Terwilliger, J . D. (1995). A powerful likelihood method for the anal-
ysis of linkage disequilibrium between trait loci and one or more
polymorphic marker loci. Am. J . Hum. Genet. 56: 777–787.
Terwilliger, J . D. (2000). A likelihood-based extended admixture
model of oligogenic inheritance in “model-based” or “model-free”
analysis. Eur. J . Hum. Genet. 8: 399–406.
Terwilliger, J . D., and Ott, J . (1992). A haplotype-based haplotype
relative risk statistic. Hum. Hered. 42: 337–346.
Terwilliger, J . D., and Weiss, K. M. (1998). Linkage disequilibrium
mapping of complex disease: Fantasy or reality? Curr. Opin. Bio-
technol. 9: 578–594.
Terwilliger, J . D., and Go ¨ring, H. H. H. (2000). Gene mapping in the
20thand 21stcenturies: Statistical methods, data analysis and
experimental design. Hum. Biol. 72: 63–132.
Wakeland, E. K., Morel, L., Mohan, C., and Yui, M. (1997). Genetic
dissection of lupus nephritis in murine models of SLE. J . Clin.
Immunol. 17: 272–281.
Watson, M. L., Rao, J . K ., Gilkeson, G. S., Ruiz, P., E icher, E . M.,
Pisetsky, D. S., et al. (1992). Genetic analysis of MRL-lpr mice:
Relationship of the Fas apoptosis gene todisease manifestations
and renal disease modifying loci. J . E xp. Med. 176: 1645–
MAGNUSSON ET AL.