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R E S E A R C H A R T I C L E Open Access
Characterization of the HLA-DRβ1 third
hypervariable region amino acid sequence
according to charge and parental
inheritance in systemic sclerosis
Coline A. Gentil
1*
, Hilary S. Gammill
1,2
, Christine T. Luu
1
, Maureen D. Mayes
3
, Dan E. Furst
4
and J. Lee Nelson
1,5
Abstract
Background: Specific HLA class II alleles are associated with systemic sclerosis (SSc) risk, clinical characteristics, and
autoantibodies. HLA nomenclature initially developed with antibodies as typing reagents defining DRB1 allele groups.
However, alleles from different DRB1 allele groups encode the same third hypervariable region (3rd HVR) sequence, the
primary T-cell recognition site, and 3rd HVR charge differences can affect interactions with T cells. We considered 3rd
HVR sequences (amino acids 67–74) irrespective of the allele group and analyzed parental inheritance considered
according to the 3rd HVR charge, comparing SSc patients with controls.
Methods: In total, 306 families (121 SSc and 185 controls) were HLA genotyped and parental HLA-haplotype origin
was determined. Analysis was conducted according to DRβ1 3rd HVR sequence, charge, and parental inheritance.
Results: The distribution of 3rd HVR sequences differed in SSc patients versus controls (p= 0.007), primarily due to an
increase of specific DRB1*11 alleles, in accord with previous observations. The 3rd HVR sequences were next analyzed
according to charge and parental inheritance. Paternal transmission of DRB1 alleles encoding a +2 charge 3rd HVR was
significantly reduced in SSc patients compared with maternal transmission (p= 0.0003, corrected for analysis of four
charge categories p= 0.001). To a lesser extent, paternal transmission was increased when charge was 0 (p=0.021,
corrected for multiple comparisons p= 0.084). In contrast, paternal versus maternal inheritance was similar in controls.
Conclusions: SSc patients differed from controls when DRB1 alleles were categorized according to 3rd HVR sequences.
Skewed parental inheritance was observed in SSc patients but not in controls when the DRβ1 3rd HVR was considered
according to charge. These observations suggest that epigenetic modulation of HLA merits investigation in SSc.
Keywords: Systemic sclerosis, Human leukocyte antigen, Skewed parental inheritance
Background
HLA class II genes contribute the largest portion of gen-
etic risk for many autoimmune diseases including sys-
temic sclerosis (SSc) [1]. The HLA DRB1 allele group
DRB1*11 has been described consistently in association
with SSc in Caucasian adults [1–3]. HLA nomenclature
evolved utilizing serological typing reagents resulting in
a“language of HLA”, with initial groupings based pri-
marily on antibody recognition of the HLA molecule.
However, the β1 chain of the HLA-DR molecule
encoded by HLA-DRB1 is characterized by three regions
of amino acid sequence hypervariability and the third
hypervariable region (3rd HVR) is thought to be the
most important site for T-cell recognition [4].
The 3rd HVR comprises amino acids 67–74 on the
alpha helix of the HLA β1 chain. Allelic variation of the
DRB1 DNA sequence can result in 3rd HVR amino acid
sequences that differ for overall 3rd HVR charge and
can impact the steric interaction between HLA mole-
cules and T cells. The importance of charge in this
region has been well demonstrated in rheumatoid arth-
ritis, where different risk-associated DRβ1 molecules
* Correspondence: cgentil@fredhutch.org
1
Division of Clinical Research, Fred Hutchinson Cancer Research Center, 1100
Fairview Ave N, Seattle, WA 98109, USA
Full list of author information is available at the end of the article
© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Gentil et al. Arthritis Research & Therapy (2017) 19:46
DOI 10.1186/s13075-017-1253-9
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
carry 3rd HVR sequences with a positive charge,
whereas a negatively charged 3rd HVR sequence is asso-
ciated with protection from the disease [5, 6].
In the current study we investigated the DRβ1 3rd
HVR, charge, and parental transmission in SSc patients
and healthy controls. We sought to examine inheritance
according to parental origin because biased HLA trans-
mission has been described in some other autoimmune
diseases, including multiple sclerosis, and in some but
not all studies of type 1 diabetes [7–9]. The primary pur-
pose of this study was to test the hypothesis that SSc risk
is modulated according to whether the mother or the
father transmitted an HLA-DRB1 allele and is impacted
by DRβ1 3rd HVR charge.
Methods
Study participants
A total of 306 unrelated families were studied, 121 in
which the proband had SSc and 185 healthy control
families. SSc patients were recruited primarily from the
Seattle, WA, USA area, with some patients from Alaska,
Montana, Oregon and other states and some identified
through the SSc family registry based in Houston, TX,
USA. Healthy controls were recruited primarily from the
Seattle, WA area. The median age at the time of sample
collection for HLA genotyping for SSc patients was
43 years (range 18–62) and for controls was 31 years
(range 4–73). SSc patients were female and controls
were predominantly female (99 female, 22 male). All
patients and controls were Caucasian. Study subjects
were included providing maternal versus paternal trans-
mission of HLA-DRB1 could be determined by HLA
genotyping studies of the patient or control and family
members. Eight SSc patients and one control were HLA
genotyped but not included in the analysis because par-
ental haplotype transmission could not be determined.
All subjects provided written informed consent. The
study was approved by the Institutional Review Board of
Fred Hutchinson Cancer Research Center.
HLA genotyping
HLA genotyping was conducted from peripheral blood,
buccal swabs, or mouthwash specimens. Genomic DNA
was extracted from whole blood or peripheral blood
mononuclear cells using the Wizard Genomic DNA
Purification Kit (Promega, Madison, WI, USA), from
mouthwash specimens using the High Pure PCR Tem-
plate Preparation Kit (Roche Diagnostics, Indianapolis,
IN, USA), or from buccal swabs using the BuccalAmp
DNA Extraction Kit (Epicentre Biotechnologies, WI,
USA). All subjects were genotyped for HLA-DRB1, as
well as for DQA1 and DQB1 loci. DNA-based typing
was conducted with the Luminex-based PCR-sequence-
specific oligonucleotide probe technique (Luminex; One
Lambda, Canoga Park, CA, USA) with alleles assigned
using HLA Fusion™3.0 standard analysis software, or
Dynal strip detection with sequence-specific oligo-
nucleotide probes (Dynal RELITM SSO, UK) followed
by identification of specific alleles by sequencing
(Applied Biosystems, Foster city, CA, USA). Amino acid
sequences were determined based on the specific alleles
and the International Immunogenetics Information
System for HLA (IGMT/HLA; www.ebi.ac.uk/ipd/imgt/
hla/allele.html).
HLA-DRβ1 3rd HVR classification
All DRB1 alleles were first categorized according to the
encoded 3rd HVR amino acid sequence, from positions 67
through 74 of the HLA-DRβ1 chain (Table 1). Among all
possible DRβ1 3rd HVR sequences, 17 were present in at
least one study subject with eight sequences present in at
least 5% of either patients or controls. Next, 3rd HVR
sequences were grouped according to their overall charge:
+2, +1, 0, −1, or −2.
In our patient and control populations no subject had a
DRB1 allele encoding a 3rd HVR sequence with a −1
charge. Therefore, our study compares four charge groups
between SSc and controls.
Table 1 HLA-DRβ1 categorized according to 3rd HVR sequences,
DRB1 alleles encoding each sequence, and associated charges
3rd HVR aa 67–74 DRB1 alleles Charge
1 LLEQRRAA 01:01, 01:02, 01:08, 04:04, 04:05,
04:08, 14:02, 14:06, 14:20
+1
2I–DE—01:03, 04:02, 11:02, 13:01, 13:02,
13:04, 13:28
−2
3—R—— 10:01 +2
4I—A—15:01, 15:02, 15:03 0
5F–D—— 11:01, 11:04, 12:02, 13:05, 16:01 0
6—D—— 16:02 0
7——K—04:01 +1
8—————E 04:03, 04:07 0
9I–D–GQ 07:01 0
10 F–R—E 09:01 +1
11 F–D—L 08:01, 08:02, 08:04, 08:06 0
12 I–D—L 08:03 0
13 F–DE—11:03 −2
14 I–D—— 12:01 0
15 ——K-GR 03:01 +2
16 I–DK—13:03 0
17 —R—E 14:01 +1
Summary is for all 3rd HVR sequences and DRB1 alleles observed in at least
one study subject
3rd HVR third hypervariable region, aa amino acids
Gentil et al. Arthritis Research & Therapy (2017) 19:46 Page 2 of 6
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Statistical analysis
After testing the normality of the distribution of con-
tinuous variables with the Shapiro–Wilk test, compari-
sons were made with the Wilcoxon rank-sum test as
appropriate. Categorical variables were compared utiliz-
ing chi-square tests or, when indicated, Fisher exact
tests. The primary outcome analyzed was maternal ver-
sus paternal inheritance of the DRβ1 3rd HVR sequence
according to charge. In addition, we compared the over-
all distribution of 3rd HVR sequences and 3rd HVR
charge in SSc patients and controls. Analysis was con-
ducted when 5% or more of patients or controls were
represented in a category and correction made for mul-
tiple comparisons by Bonferroni adjustment of the
threshold for significance. Logistic regression was carried
out adjusting for sex because some controls were male,
but this revealed no differences from the unadjusted
associations.
Because a parent of origin effect has not been studied
previously in SSc, a precise estimate of statistical power
is precluded. However, our available sample size of 121
cases and 185 controls is estimated to provide more than
90% power to detect a two-fold difference in parental
origin from an expected 50/50 in controls to 25/75 in
cases, assuming α= 0.05 and a two-tailed test.
Results
Among SSc patients, 64% had diffuse SSc and 36% lim-
ited SSc (77 and 44 respectively). Antibodies to topo-
isomerase I (ATA) were positive in 34% of patients (37
of 109 tested) and to centromere (ACA) in 19% of pa-
tients (17 of 90 tested). The median age of SSc onset
was 37 years, range 16–58.
HLA-DRB1 alleles were classified based on the amino
acid sequence of the encoded 3rd HVR (amino acids
67–74) and the overall charge of each unique 3rd HVR se-
quence was determined (Table 1). Comparing SSc patients
with healthy controls, the overall distribution of 3rd HVR
sequences was significantly different (p=0.007) (Table 2).
The sequence “FLEDRRAA”(sequence category 5 in
Table 1) was significantly enriched in SSc patients com-
pared with controls (p=0.004, pvalue corrected for mul-
tiple comparisons p
c
= 0.032), primarily due to enrichment
of DRB1*11:04 among SSc patients, as reported previously
[3]. The sequence “FLEDRRAL”(sequence category 11 in
Table 1) was also enriched in SSc patients (p=0.006,p
c
=
0.048) (Table 2), due to some increase of DRB1*08:01 and
DRB1*08:02 in SSc patients. These observations are con-
cordant with a previous report of an increase in the motif
“FLEDR”in a French population [10]. In contrast to the
French study, however, we did not see enrichment of this
sequence encoded by DRB5, which instead was decreased
in our population (data not shown).
Overall, the 3rd HVR charge on SSc haplotypes was
similar to that of controls; of the 242 SSc and 370 con-
trol haplotypes respectively, 11% and 12% had a +2
charge, 26% and 31% had a +1 charge, 50% and 44% had
a charge of 0, and 13% and 13% had a −2 charge. As
noted previously, no 3rd HVR sequence represented in
our populations carried a −1 charge. Thirty-six percent
of SSc patients and 34% of controls carried the same
charge on both parental haplotypes. When considering
both haplotypes together, 19%, 32%, and 49% of the SSc
patients had overall negative, neutral, or positive charge
respectively, compared with 20%, 22%, and 58% of
healthy controls (Fig. 1).
Analysis was conducted considering maternal or pater-
nal DRB1 allele inheritance according to the charge of
the DRβ1 3rd HVR. The overall distribution of inherit-
ance indicated significant skewing among SSc patients
(p< 0.001). Among SSc patients the father transmitted a
3rd HVR sequence with a +2 charge significantly less
often than the mother (p= 0.0003). Almost 90% of the
time, transmission was maternal rather than paternal
(Fig. 1). This skewed parental inheritance remained sig-
nificant (p= 0.001) when corrected for comparing four
different categories of charge. The skewed inheritance
Table 2 Allele frequencies classified according to the third
hypervariable region in controls and SSc patients
Controls SSc
HVR region n(%) n(%) pvalue OR (95% CI)
1 68 (18.4) 34 (14.0) 0.160 0.73 (0.46–1.14)
2 44 (11.9) 24 (9.9) 0.447 0.82 (0.48–1.38)
3 3 (0.8) 1 (0.4)
4 58 (15.7) 21 (8.7) 0.012 0.51 (0.30–0.87)
5 36 (9.7) 43 (17.8) 0.004
§
2.00 (1.25–3.23)
6 0 (0.0) 1 (0.4) –
7 34 (9.2) 25 (10.3) 0.640 1.14 (0.66–1.96)
8 4 (1.1) 8 (3.3)
9 48 (13.0) 29 (12.0) 0.718 0.91 (0.56–1.49)
10 4 (1.1) 2 (0.8)
11 8 (2.1) 16 (6.6) 0.006
#
3.20 (1.35–7.61)
12 1 (0.3) 0 (0.0) –
13 5 (1.3) 6 (2.5)
14 6 (1.6) 4 (1.7)
15 41 (11.1) 26 (10.7) 0.896 0.97 (0.57–1.63)
16 3 (0.8) 0 (0.0) –
17 7 (1.9) 2 (0.8)
All 370 242
Third HVR overall distribution, SSc patients versus controls, p= 0.007
§
p
c
= 0.032
#
p
c
= 0.048
SSc systemic sclerosis, HVR hypervariable region, OR odds ratio, CI confidence
interval, p
c
pvalue corrected for multiple comparisons
Gentil et al. Arthritis Research & Therapy (2017) 19:46 Page 3 of 6
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
was observed among patients with both diffuse and lim-
ited SSc and was not different for those with younger
versus older age of SSc onset. Skewed inheritance for
overall distribution, and for each charge category, was
not observed in the control population in which inherit-
ance was not different from 50–50, as expected. To a
lesser extent, 3rd HVR sequences with a 0 charge were
inherited more often from the father than the mother
among SSc patients (p= 0.021, p
c
= 0.084). Parental
transmissions of 3rd HVR sequences with +1 or −2
charge were not significantly skewed (Fig. 1).
While our study was designed to test a hypothesis
regarding charge of the DRβ1 3rd HVR sequence and
parental inheritance, linkage disequilibrium of DRB1 is
very strong with DQA1 and DQB1, and secondary ana-
lysis was conducted for these loci. DQβ1 has a similarly
located hypervariable region from amino acids 70
through 77. DQB1 alleles represented in our study popu-
lations had an overall net charge of +3, +1, 0, −1, or −2
(Table 3). Not surprisingly, the +3 charge for DQβ1was
also significantly decreased for paternal versus maternal
transmission, because DRB1*03:01 (+2 DRβ1 3rd HVR)
haplotypes in our populations with few exceptions car-
ried DQB1*02:01, which has a +3 charge. Patterns of
linkage disequilibrium did not permit clear separation of
the role of DQB1 from DRB1. However, the skewing for
DQβ1 with a +3 charge was less pronounced than that
described for DRβ1; eight were transmitted paternally
versus 31 transmitted maternally. Additionally,
DRB1*07:01 haplotypes can carry either DQB1*02:02
(+3) or DQB1*03:03 (0), and there was no suggestion of
skewed paternal inheritance for these two haplotypes or
an increase in DRB1*07:01–DQB1*02:02 haplotypes in
the SSc population overall (Additional file 1: Table S1).
Polymorphisms on DQα1 are distributed somewhat
differently from DRβ1 and DQβ1, but considered for
amino acids 47–56 the net charge was +4, +2, or +1 for
alleles represented in our study population and did not
differ due to parental inheritance (Table 4). This result
was also consistent with linkage disequilibrium in the
HLA class II region, but results for DQA1 could be
Fig. 1 Distribution of HLA-DRβ1 3rd HVR sequences according to charge and parental inheritance. Colors represent the overall 3rd HVR charge for
each (biallelic) individual, considering both maternal and paternal haplotypes; red, overall charge was negative; yellow, overall charge was neutral; and
green, overall charge was positive. Among SSc patients there was marked skewing of parental inheritance when the 3rd HVR sequence carried a +2
charge (p= 0.0003, p
c
= 0.001) whereas controls showed a similar frequency of inheritance from either parent, as would be expected. Skewing was also
observed, to a lesser extent, among SSc patients when the 3rd HVR sequence carried a 0 charge (p=0.021,p
c
= 0.08). Columns and rows with a −1
charge are 0 because no study subject had a 3rd HVR sequence with this charge (Color figure online). SSc systemic sclerosis
Table 3 DQβ1 hypervariable region and charge, amino acids
70–77
a
Amino acid position Overall charge
Allele 70 71 72 73 74 75 76 77
05:01/2/3 G A R A S V D R +1
06:01/4/9/11,
03:01/2/3
RT––E––T0
06:02/3/11/16 –T––EL–T−1
02:01/2 RK––A–––+3
b
04:01/2 ED–––––T−2
a
Amino acids that differ from the consensus (alleles on the first line) by charge
are presented in bold. Sequences encoded by uncommon alleles not present
in our study populations are not included
b
Among SSc patients with DQβ1 + 3 charge, eight were inherited paternally
and 31 inherited maternally (pcorrected = 0.001)
Table 4 DQα1 hypervariable region and charge, amino acids
47–56
a
Amino acid position Overall charge
b
Allele 47 48 49 50 51 52 53 54 55 56
01:01/2/3
/4/5
RWPEFSKFGG+1
02:01 K ––L–HR–R–+4
03:01/2/3 Q––L–RR–RR+4
04:01 C––V–RQ–R–+2
05:01/3/5 C––V–RQ–R–+2
06:01 C––V–RQ–R–+2
a
Amino acids that differ from the consensus (alleles on the first line) by charge
are presented in bold. Sequences encoded by uncommon alleles not present
in our study populations are not included
b
Parental inheritance was not skewed according to any DQα1 charge category.
Similarly, results were not skewed if alternatively considered to extend to
include a polymorphism at DQα1 amino acid 64
Gentil et al. Arthritis Research & Therapy (2017) 19:46 Page 4 of 6
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distinguished from DRB1 because multiple different
DRB1 alleles are on haplotypes with the similar DQA1
alleles and some DQA1 alleles other than DQA1*05:01
also encode a +2 charge.
As expected because of linkage disequilibrium, the
DRB1*03:01–DQA1*05:01–DQB1*02:01 haplotype was
similarly skewed with three inherited paternally versus 21
inherited maternally (p
c
= 0.001), although again skewing
was somewhat less pronounced than when considered for
DRβ1. (Haplotypes that were common in our study popu-
lation are presented in Additional file 1: Table S1.)
Discussion
In the current study we investigated patients with SSc
and healthy controls, analyzing the DRβ1 3rd HVR
according to charge irrespective of specific DRB1 alleles
and considered parental inheritance. The importance of
the DRβ1 3rd HVR and of charge in this region is well
established in another autoimmune disease, rheumatoid
arthritis, for which underlying HLA disease susceptibility
is believed to be due to amino acid motifs of the DRβ1
3rd HVR that carry a similar charge [5, 6]. Our primary
interest was to ask whether SSc patients differed from
healthy controls if considered for parental origin of the
DRβ1 3rd HVR categorized according to charge. In con-
trast to healthy individuals among whom parental inher-
itance was 50–50 as expected, we found significant
skewing of parental inheritance in SSc patients. The
most striking finding was that the father transmitted a
DRB1 allele encoding a +2 charge 3rd HVR significantly
less often than the mother in SSc patients. With respect
to DRB1 allele frequencies, our study population was simi-
lar to prior reports of similar populations [1–3], with the
patient population primarily enriched for DRB1*11:04
compared with controls.
Skewed parental inheritance of HLA-DRB1 alleles in
SSc strongly suggests a parent-of-origin effect. Parent-
of-origin effects are well described for a number of genes
and are especially well recognized as important in early
development [11]. Skewing from the expected random
(50–50) inactivation of maternal versus paternal X-
chromosomes has been reported previously in women
with SSc, as well as in women with rheumatoid arthritis
and some other diseases [12, 13]. A few studies have
evaluated parental inheritance of HLA in other auto-
immune diseases. In type 1 diabetes, parent-of-origin
skewing in HLA inheritance has been described in some,
but not all, studies [8, 9]. In patients with multiple scler-
osis, HLA transmission was distorted by the parent of
origin and by gender of the affected offspring [7]. A
differential methylation signal with a peak at HLA-DRB1
was observed in CD4
+
T cells of multiple sclerosis pa-
tients compared with controls in another study, suggest-
ing a potential explanation for this observation [14].
A number of phenomena can underlie a parent-of-
origin effect. The best characterized parent-of-origin ef-
fect is that associated with genomic imprinting [11]. Em-
bryonic lethality is one explanation for a parent-of-origin
effect and, although skewing was not observed in our
healthy population, it is possible that an extreme pheno-
type could result in loss of embryos destined to develop
SSc in later life. The rate of decay for small molecules or
epigenetic marks in sperm and ova affecting survival to
gametogenesis has also been hypothesized to play a role
in distorted haplotype transmission [7]. Additionally, the
molecules CCCTC-binding factor (CTCF) and Class II
transactivator (CIITA), two units of a transcription
complex involved in HLA class II gene transcription, are
thought to be subject to epigenetic modifications [15,
16]. The current results are of further interest in light of
a recent study that implicated fetal programming in SSc
[17] as well as increasing evidence for a role of epigen-
etic regulation in SSc pathogenesis [18].
There are a number of limitations to our study. SSc
patients in our study were all female and it would be of
additional interest to know whether males and/or chil-
dren with SSc differ. Some of our controls were male
and younger than the patient population. However, nei-
ther would be anticipated to affect the overall study ob-
servations because controls had the expected random
(50–50) distribution regardless of gender or age. SSc is
also a rare disorder offsetting any potential bias due to
development of SSc later in life by a younger control.
Another limitation is that all of our study subjects were
Caucasian because too few patients from other racial/
ethnic backgrounds were available for analysis. Also, the
number of SSc patients was modest and a larger study
would be needed to evaluate potential differences ac-
cording to clinical and autoantibody characteristics. An-
other limitation is that alleles carrying a +2 charge other
than DRB1*03:01 were uncommon in our population so
the skewed inheritance observed could be specific to genes
on the DRB1*03:01 haplotype, including DQB1*0201,
rather than the DRβ1 3rd HVR +2 charge. It should also be
added that some amino acids are subject to posttransla-
tional modification that results in a change of charge [19]
and, while this has largely been examined on autoantigens,
HLA molecules are themselves also presented as self-
peptides by other HLA molecules [20].
Conclusions
Skewed parental HLA inheritance was observed among
SSc patients compared with healthy controls. Paternal
transmission was significantly reduced compared with
maternal transmission when the HLA-DRB1 allele
encoded a 3rd HVR with a +2 charge and to a lesser ex-
tent increased when charge was 0, in SSc patients but
not in controls. To our knowledge, skewed parental
Gentil et al. Arthritis Research & Therapy (2017) 19:46 Page 5 of 6
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
inheritance of HLA-DRB1 alleles has not been reported
previously in SSc. Provided our results are replicated,
future investigations into the underlying mechanism(s)
for a parent-of-origin effect of HLA inheritance in SSc
may lead to new insight into the role of HLA molecules
in SSc pathogenesis.
Additional file
Additional file 1: Table S1. DRB1-DQA1-DQB1 common haplotypes in
SSc patients and controls. (DOCX 44 kb)
Abbreviations
HLA: Human leukocyte antigen (or allele); HVR: Hypervariable region;
SSc: Systemic sclerosis
Acknowledgements
The authors are grateful to The Scleroderma Family Registry and to all of the
patients and family members as well as the healthy controls and their family
members for their participation in this study and to Judy Allen MPH and
Samantha Bell MPH for study coordination. They thank Alex Forsyth for
additional review of HLA alleles and amino acid sequences.
Funding
This work was supported by NIH grant RO1 AI-41721 and a grant from The
Scleroderma Foundation.
Availability of data and materials
The datasets used and analyzed in the current study are not part of a public
database but are available from the corresponding author upon reasonable
request.
Authors’contribution
CAG and CTL conducted experiments. CAG, HSG, and JLN analyzed and
interpreted the data. MDM and DEF contributed patients and clinical
assessments and contributed to the summary of results. CAG and JLN
primarily wrote the manuscript. JLN conceived the study. All authors
approved the final manuscript.
Competing interests
The authors declare that they have no competing interests.
Consent for publication
Not applicable.
Ethics approval and consent to participate
All subjects provided written informed consent. The study was approved by
the Institutional Review Board of Fred Hutchinson Cancer Research Center.
Author details
1
Division of Clinical Research, Fred Hutchinson Cancer Research Center, 1100
Fairview Ave N, Seattle, WA 98109, USA.
2
Department of Obstetrics and
Gynecology, University of Washington, Seattle, WA, USA.
3
Division of
Rheumatology and Clinical Immunogenetics, University of Texas Health
Science Center at Houston, Houston, TX, USA.
4
Division of Rheumatology,
University of California, Los Angeles, CA, USA.
5
Division of Rheumatology,
University of Washington, Seattle, WA, USA.
Received: 3 November 2016 Accepted: 1 February 2017
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