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Variation in the relative copy number of the TLR7 gene in patients with systemic lupus erythematosus and healthy control subjects.

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ARTHRITIS & RHEUMATISM
Vol. 56, No. 10, October 2007, pp 3375–3378
DOI 10.1002/art.22916
© 2007, American College of Rheumatology
Variation in the Relative Copy Number of
the TLR7 Gene in Patients With
Systemic Lupus Erythematosus and Healthy Control Subjects
James Kelley, Martin R. Johnson, Graciela S. Alarcón, Robert P. Kimberly,
and Jeffrey C. Edberg
Objective. To determine whether there is an increase in the number of TLR7 gene copies in patients
diagnosed as having systemic lupus erythematosus
(SLE) and whether gene amplification influences the
autoantibody profiles in SLE patients, as has recently
been reported in the BXSB/Yaa mouse model of
lupus.
Methods. We used a modified real-time quantitative polymerase chain reaction protocol to calculate the
relative TLR7 gene copy number according to the comparative 2–⌬⌬Ct method in 99 SLE patients and 91
healthy controls matched for sex and ethnicity. Autoantibody profiles were determined by standard methods.
Results. The relative number of TLR7 gene copies
in SLE patients and healthy controls varied; however,
no significant concordance between the number of relative gene copies and the SLE phenotype was found.
There was also no difference in variation by ethnic
group. Comparison of the relative gene copy numbers
according to the presence or absence of antinuclear
antibodies (ANAs), the ANA staining patterns, and the
presence or absence of anti-RNA–associated antigen
antibody showed no statistically significant difference in
the SLE patients.
Conclusion. We determined that although the
relative gene copy number of TLR7 varied in both SLE
patients and healthy controls, it was not significantly
increased among our SLE patients as compared with
our controls. We found no detectable trend for an
association between the relative gene copy number and
the autoantibody profile in SLE patients.
A recent article in Science (1) reported that a
genomic segmental duplication, which included the murine Toll-like receptor 7 (Tlr7) gene, and the translocation of this segment to the Y-linked autoimmune accelerator (Yaa) locus were associated with autoreactive B
cell responses to RNA-related antigens. Similar findings
of a duplication of the Yaa locus have been independently reported (2). The Yaa locus has previously been
shown to increase the severity of lupus-like disease in
males of the BXSB mouse strain (3) and to change their
autoantibody specificity (4), leading to the suggestion
that increased expression of Tlr7 due to this increase in
genomic DNA may affect the autoimmune phenotype of
these mice.
Mouse models can provide important insights
into human immune function and disease; however, the
mechanisms require careful validation, since many
known immunologic differences exist between the two
species (5). A role of TLR7 in humans with systemic
lupus erythematosus (SLE) is consistent with its ability
to induce the release of interferon-␣ (IFN␣), a cytokine
that has been shown to be increased in the serum of
patients with SLE (6). Since TLR7 is located in a
syntenic region of the X chromosome in humans and
mice and since an increased prevalence of SLE in
women (7) suggests an X-linked genetic component, we
sought to determine whether, similar to the findings in
the Yaa mouse, there were increased gene copy numbers
of TLR7 in humans with SLE.
Supported by an NIH Program Project grant in the Genetics
of SLE (P01-AR-49084). Dr. Kelley’s work was supported by a grant
from the NIH Training Program in Rheumatic Diseases Research
(T32-AR-07450).
James Kelley, PhD, Martin R. Johnson, PhD, Graciela S.
Alarcón, MD, MPH, Robert P. Kimberly, MD, Jeffrey C. Edberg,
PhD: The University of Alabama at Birmingham.
Address correspondence and reprint requests to Jeffrey C.
Edberg, PhD, Department of Medicine, Division of Clinical Immunology and Rheumatology, The University of Alabama at Birmingham,
1530 Third Avenue, Shelby Interdisciplinary Research Building 207,
Birmingham, AL 35294. E-mail: jedberg@uab.edu.
Submitted for publication October 18, 2006; accepted in
revised form June 22, 2007.
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KELLEY ET AL
Figure 1. Relative number of copies of the TLR7 gene in genomic DNA from male and female patients with systemic lupus erythematosus (SLE)
and healthy controls. Relative gene copy number of TLR7 in genomic DNA refers to the ratio of the quantity of TLR7 produced in a quantitative
polymerase chain reaction (PCR) compared with that of a housekeeping gene, either A, GAPDH or B, HPRT1, as calculated by the 2–⌬⌬Ct method.
Each sample was compared with 1 sample that was selected as a calibrator, to allow comparison of the relative amounts between individuals;
therefore, these values reflect the comparison of the relative amount and not the absolute copy number. Given that the amount of resulting PCR
product corresponds to the amount of genomic template DNA in each subject, the data presented here show no difference in the quantitative level
of TLR7 in genomic DNA from SLE patients and healthy controls. Normalization against a housekeeping gene corrects for any sample-to-sample
variation in the amount of template genomic DNA present in the PCR product. Symbols represent individual subjects. Bars show the mean ⫾ SD
for each group.
PATIENTS AND METHODS
The relative number of copies of the TLR7 gene in
genomic DNA from 50 Caucasian and 49 African-American
patients with SLE (55 women and 44 men) along with 91 sexand ethnicity-matched healthy control subjects was determined
using a modified real-time quantitative polymerase chain
reaction (PCR) method similar to that previously described for
the detection of copy number polymorphisms in an immunologically related gene family (8). Samples were collected at The
University of Alabama at Birmingham. All subjects gave their
informed consent, and the Institutional Review Board approved the study.
The SLE patients met the revised and updated criteria
established by the American College of Rheumatology (9,10).
We collected laboratory clinical data from as early as 1998,
when available, on 63 of our 99 SLE patients, which included
results of tests for anti-DNA antibodies, anticardiolipin antibodies, antinuclear antibodies (ANAs; including reactivity
pattern, such as speckled, homogeneous, or nucleolar), and
RNA–associated antigen autoantibodies (including anti-SSA/
Ro, anti-SSB/La, anti-Sm, and anti-RNP).
We performed PCR quantitations in quadruplicate for
each of the 190 samples, using Assay-by-Design TaqMan
primers and FAM-labeled minor groove binder probes (all
from Applied Biosystems, Foster City, CA) for TLR7
(Xp22.3). Sequences were as follows: for the forward primer,
5⬘-CAGTATTGTGCTGTCTTTGAAATGTAAA-3⬘; for the
reverse primer, 5⬘-TGGGCCCAATAGCATCAACT-3⬘; and
for the probe, 5⬘-TTGATGTCTTCTCTTTCTC-3⬘. To ensure
that only genomic DNA was detected, the primers were
designed to span an intron–exon border, and each sample was
treated with an RNA-degrading enzyme during the DNA
extraction process. We controlled for differences in DNA
concentration between samples by normalizing against hypoxanthine phosphoribosyltransferase 1 (HPRT1; Xq26.1) and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 12p13),
both of which have been previously reported to be housekeeping genes (11).
Reactions were conducted on the ABI 7900HT system,
and calculations were performed using the 2–⌬⌬Ct method
(12,13). Briefly, this method calculates the difference in cycle
thresholds (the number of PCR cycles required to produce a
set of fixed thresholds) between the gene of interest and a
housekeeping gene (⌬Ct). Subsequent calculations normalize
the ⌬Ct of each sample to a calibrator that is assigned a relative
expression value of 1.00 (⌬⌬Ct). Assuming that the amount of
PCR product doubles with each successive PCR cycle, calculating the 2–⌬⌬Ct value will provide the relative amount of DNA
initially available for amplification in each quantitative PCR
run. Therefore, the 2–⌬⌬Ct method reveals differences in the
relative gene copy numbers between the samples tested (12). A
range for each expression value was calculated based on the
standard deviation(s) of the ⌬⌬Ct value, where 2⫺(⌬⌬Ct ⫹ s) is
the lower limit and 2–(⌬⌬Ct ⫺ s) is the upper limit. All statistical
TLR7 GENE COPY NUMBER VARIATIONS IN PATIENTS WITH SLE
measures were calculated with GraphPad Prism 4.03 software
(GraphPad, San Diego, CA).
RESULTS
Although the relative number of copies of the
TLR7 gene in patients with SLE and in healthy control
subjects varied, no significant concordance between the
relative number of gene copies and the SLE phenotype
was found. There was also no difference in variation by
ethnic group. Therefore, we conclude that there is not an
increase in relative copy number of the TLR7 gene in
our population of SLE patients as compared with our
population of controls (Figure 1).
Correlational analysis (P ⬍ 0.0001) of both
housekeeping genes, which were independently used in
this study to normalize the amount of DNA in each
sample, demonstrated that either gene could be used
separately to calculate relative expression levels of
TLR7. We are also confident in the technical precision
of this method because the average variation in cycle
3377
number for our PCR results, which were performed in
quadruplicate for each of the 3 assays (TLR7, GAPDH,
and HPRT1) on each of the 190 samples, was ⬍1%
(0.86%) in this measure of intraassay precision (13).
Additionally, when this study was repeated on a selection of 22 samples and then a selection of 54 samples (27
with SLE and 27 controls) using all 3 assays on different
days, there was a significant correlation between samples
for the interassay data (P ⫽ 0.0016) (13).
Since the Tlr7 copy number in mice has been
shown to influence the titer and pattern of antibodies
against nuclear antigens (1), we additionally attempted
to correlate the relative gene copy number of TLR7 with
autoantibody presence, titer, and pattern. Stratifying the
relative gene copy number results by both positive and
negative ANA titers, by ANA patterns, and by anti–
RNA–associated antigen antibody presence showed neither a statistically significant difference nor an unequal
distribution. This finding led us to conclude that the
relative gene copy number of TLR7 does not have a
significant pattern or relationship to the autoantibody
profile in patients with SLE (Figure 2).
DISCUSSION
Figure 2. Autoantibody profile versus the relative TLR7 gene copy
number normalized against HPRT1 in systemic lupus erythematosus
patients, by antinuclear antibody (ANA) positivity (in a speckled or
homogeneous fluorescence staining pattern), ANA negativity, RNAassociated antigen (RNA-AA) autoantibody (anti-SSA/Ro, anti-SSB/
La, anti-Sm, and anti-RNP) positivity, and RNA-AA negativity, and in
healthy controls. To eliminate any variation due to sex, only data for
the relative copy number of the TLR7 gene normalized against HPRT1
were used in this analysis. The distribution of each pattern among the
range of relative gene copy numbers presents no significant relationships, as determined by analysis of variance (P ⫽ 0.7514). Symbols
represent individual subjects. Bars show the mean ⫾ SD for each
group.
While Pisitkun and colleagues (1) demonstrated
an example of copy number variation in a gene that
influenced a complex disease in mice, their specific
finding of a genomic increase in Tlr7 in a murine model
of lupus cannot be translated directly to humans with
SLE. Our findings in SLE patients do not preclude a role
for TLR7 genetic variants, since coding region and/or
regulatory variants (single-nucleotide polymorphisms)
in the TLR7 gene are largely unexplored. In addition, an
increase in the amount of message RNA for TLR7 may
influence SLE.
We detected variations in the relative copy number of the TLR7 gene among a portion of both the
patient and controls samples tested in our study. Due to
the presence of this variation in both groups, we could
not statistically associate this observed structural variation directly with the SLE phenotype. However, given
the complex genetic nature of this autoimmune disease
and given that TLR7 has potential functional relevance
to SLE (6), we cannot rule out the possibility that copy
number variations in TLR7 may influence the genetic
background susceptibility for SLE. Since additional genetic variants are associated with lupus-like disease in
mice, along with a TLR7 gene copy number variation
(1), such a variant in the presence of other genetic
3378
KELLEY ET AL
factors may have a contributive, additive effect on the
human SLE phenotype in a subset of patients.
Another recent study has emphasized sex differences in TLR7 function that might influence the SLE
phenotype. Peripheral blood lymphocytes (PBLs) from
healthy women release more IFN␣ after TLR-7 stimulation as compared with PBLs from healthy men, an
effect that was not seen after stimulation with TLR-9,
another inducer of IFN␣ (14). The increase in IFN␣
among PBLs from women was not due to a defect in X
chromosome inactivation (14). Our results validate the
findings of that study, since a significant difference in the
relative TLR7 gene copy number in genomic DNA
normalized against GAPDH was observed between men
and women (P ⫽ 0.0138). While TLR7 may influence the
genetic background of SLE pathogenesis and contribute
to the difference in disease prevalence between the
sexes, such a contribution in humans cannot be directly
attributed to an increase in gene copy number in a
standardized quantity of genomic DNA as was seen in
the Yaa mouse.
ACKNOWLEDGMENTS
We would like to thank Jan Dumanski for critical
review of the manuscript, Amy Peterson for technical assistance, and S. Louis Bridges for use of the ABI 7900HT
sequencer.
AUTHOR CONTRIBUTIONS
Dr. Kelley had full access to all of the data in the study and
takes responsibility for the integrity of the data and the accuracy of the
data analysis.
Study design. Kelley, Johnson, Kimberly, Edberg.
Acquisition of data. Kelley, Alarcón.
Analysis and interpretation of data. Kelley, Johnson, Alarcón, Kimberly, Edberg.
Manuscript preparation. Kelley, Edberg.
Statistical analysis. Kelley, Edberg.
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