Download the role of germline polymorphisms in the t-cell

British Journal of Rheumatology 1998;37:454–458
THE ROLE OF GERMLINE POLYMORPHISMS IN THE T-CELL RECEPTOR IN
SUSCEPTIBILITY TO ANKYLOSING SPONDYLITIS
M. A. BROWN, M. RUDWALEIT,* K. D. PILE,† L. G. KENNEDY,‡ J. SHATFORD, C. I. AMOS,§
K. SIMINOVITCH,¶ L. RUBIN,d A. CALIN‡ and B. P. WORDSWORTH
Wellcome Trust Centre for Human Genetics, Windmill Road, Headington, Oxford, *Rheumatology Section, Department of
Endocrinology and Nephrology, University Hospital Benjamin Franklin, Berlin, Germany, †Queen Elizabeth Hospital, Adelaide,
Australia, ‡Royal National Hospital for Rheumatic Diseases, Upper Borough Walls, Bath, §Department of Epidemiology,
University of Texas M.D. Anderson Cancer Center, Houston, TX, USA, ¶Mount Sinai Hospital, Suite 656A, 600 University
Avenue, Toronto and dWomen’s College Hospital, University of Toronto, Toronto, Ontario, Canada
SUMMARY
The role of germline polymorphisms of the T-cell receptor A/D and B loci in susceptibility to ankylosing spondylitis was
investigated by linkage studies using microsatellite markers in 215 affected sibling pairs. The presence of a significant
susceptibility gene ( lambda 1.6) at the TCRA/D locus was excluded (LOD score < − 2.0). At the TCRB locus, there was
weak evidence of the presence of a susceptibility gene (P = 0.01, LOD score 1.1). Further family studies will be required to
determine whether this is a true or false-positive finding. It is unlikely that either the TCRA/D or TCRB loci contain genes
responsible for more than a moderate proportion of the non-MHC genetic susceptibility to ankylosing spondylitis.
K : T-cell receptor, Ankylosing spondylitis, Spondyloarthritis, Genetics, Linkage, Microsatellite, Polymorphism.
T aetiology of ankylosing spondylitis (AS) is
unknown, but a combination of genetic and environmental factors is probably involved. Evidence from
twin and transgenic animal studies suggests that the
environmental trigger for the disease is ubiquitous, and
that genetic factors are the main determinants of
liability towards the disease [1]. A direct role for HLAB27 in susceptibility to AS is suggested by the strong
association and linkage of this gene with AS [2], and
from B27-transgenic mouse and rat experiments [3, 4].
Other HLA-B genes, such as B60, have also been
implicated in susceptibility to the disease [5]. The
presence of susceptibility genes for AS outside the
MHC is supported by family and twin studies which
demonstrate that genetic susceptibility to AS is likely
to be due to the effect of multiple loci acting epistatically [1, 6 ]. Even in B27-transgenic rats, the background strain of the rat influences the penetrance of
the arthritis [7]. The contributions of B27 alone, and
of MHC genes collectively, have been calculated to be
50% or less of the total genetic contribution to the
disease [1, 2, 8]. The genes responsible for the nonMHC component of genetic susceptibility to AS are
unknown. A small number of candidate genes have
been investigated by linkage and case–control studies,
but no reported association has ever been replicated
(reviewed in [9]).
Since the primary function of HLA-B molecules is
the presentation of peptide antigens to lymphocytes, it
has been suggested that HLA-B27-restricted peptide
presentation to cytotoxic T lymphocytes (CTL) is
important in the pathogenesis of AS. The presence of
CD4 and CD8 B27-restricted CTL in synovial fluid
from patients with AS and reactive arthritis has been
demonstrated [10, 11]; therefore, genetic determinants
of the function of CTL may influence susceptibility
to AS.
The a and b components of the T-cell receptor
( TCR), which interact directly with the HLA–peptide
complex, are encoded by genes on chromosomes 14
( TCRA at 14q11.2) and 7 ( TCRB at 7q35), respectively. The TCRD locus, which codes for the d chain
of cd T cells, also lies within the TCRA locus. The
germline code of the TCRA and B loci consists of V
(variable), J ( junctional ) and C (constant) regions,
with the addition of a D (diversity) region in the TCRB
locus. Somatic rearrangement of these gene segments
produces contiguous V/J/C or V/D/J/C genes coding
for the TCR. Further TCR diversity results from the
addition of non-germline-coded nucleotides at gene
segment junctions (N region diversity). The TCRB
locus has recently been fully sequenced, revealing 65
VB gene segments which can be divided into 30 subfamilies with 75% sequence homology [12]. There are
thought to be at least 100 VA gene segments [13]. The
germline sequences of the variable gene segments
themselves have high mutation rates [12]. Sequence
variation within the TCR loci has arisen from singlebase substitutions, small insertions or deletions, and
duplication or deletion of one or more members of
tandem arrays of repeat segments. Sequence investigation of the TCRA/D locus has found on average
one polymorphism every 433 base pairs, which is
greater than elsewhere in the genome where polymorphisms are estimated to occur once in every
500–1500 base pairs [14]. Some sequence variations
occur with such high frequency as to suggest that they
offer a selective advantage, such as the loss of autoimmune tendencies [12], or resistance to infection.
Clearly, the opposite effect could also result with
Submitted 19 August 1997; revised version accepted 14 October
1997.
Correspondence to: M. Brown, Wellcome Trust Centre for Human
Genetics, Windmill Road, Headington, Oxford OX3 7BN.
© 1998 British Society for Rheumatology
454
BROWN ET AL.: TCR POLYMORPHISMS AND SUSCEPTIBILITY TO AS
germline polymorphisms increasing the risk of
autoimmunity.
The relevance of polymorphisms to disease predisposition can be studied by association or linkage
studies. Association studies compare the frequency of
known polymorphisms in patients and controls.
Positive findings suggest an effect on disease susceptibility either from the polymorphism studied, or
a further polymorphism in the surrounding region
which is in linkage disequilibrium. The large number
of potential candidate polymorphisms within the
TCRA/D and TCRB loci, the lack of sequence data
for most of them, and the weakness of linkage disequilibrium at a population level at these loci would make
a comprehensive study of these loci by association
methods extremely difficult. Linkage studies do not
require prior knowledge of the exact nature of the
disease causing mutations and therefore may be more
powerful in the analysis of such regions.
Association studies of TCR polymorphisms in
rheumatoid arthritis, another inflammatory arthropathy with strong HLA associations, have given conflicting results. In the largest study reported to date,
Cornélis et al. [15] have suggested a weak but significant effect (odds ratio 1.3, P = 0.008) from TCRAV8S1
in a case–control study of 800 patients and matched
controls. We have recently excluded the presence of a
major susceptibility gene at either the TCRA/D or
TCRB loci in rheumatoid arthritis [16 ]. The aim of
the current study was to assess the role of germline
polymorphisms of the TCRA and TCRB loci in susceptibility to AS.
METHODS
Patients and controls
Affected AS sib pairs were recruited from the outpatient clinics of rheumatology departments in the UK
and from the Royal National Hospital for Rheumatic
Disease (RNHRD) Ankylosing Spondylitis Database.
Eight additional families recruited from Newfoundland
have been reported elsewhere [2]. All patients fulfilled
the modified New York Criteria for AS [17]. Complete
and incomplete nuclear families were studied; additional unaffected siblings were recruited where available when samples could not be obtained from parents.
All patients had their diagnosis confirmed by a rheumatologist and had radiographic evidence of sacroiliitis.
Ethical approval for the study was granted by the
Central Oxford Research Ethics Committee and the
University of Toronto Ethics Review Panel. A total of
215 affected sib pairs from 175 families were used
in the linkage study. DNA samples were available
from 820 individuals, of whom 449 had AS. The
families included 28 one-generation families, 122 twogeneration families, 22 three-generation families and
three four-generation families.
Peripheral venous blood samples were obtained and
DNA extracted by the standard phenol/chloroform/
proteinase K method.
Three microsatellites spanning the TCRA and TCRB
loci were used. For TCRA, these were D14S50, TCRA
455
and D14S64 (D14S50 and D14S64 lie 2.9 and 8 cM
either side of TCRA, respectively). For TCRB, these
were D7S509, TCRVb6.7 and D7S688 (D7S509 and
D7S688 lie 8.2 cM on either side of TCRVb6.7).
Forward polymerase chain reaction (PCR) primers
were 5∞ labelled with either FAM (D7S688, D14S50,
D14S64, TCRA) or TET (D7S509, TCRVb6.7) fluorescent dyes. The reaction mix used was 1 ml of 10 ×
NH4 buffer, 200 m of each dNTP, 11.4 ng of each
primer, 0.2 U of Taq DNA polymerase, magnesium
chloride (1.5–3.5 m) and sterile distilled water to
a volume of 10 ml. The cycling conditions for each
primer were 94°C for 1 min, annealing for 1 min,
elongation 72°C for 45 s. ‘Omnigene’ thermal cyclers
(Hybaid, Teddington) were used. Optimal magnesium
concentrations, annealing temperatures and cycle
numbers for each primer are given in Table I.
Electrophoresis was performed using ABI 373 DNA
semiautomated sequencers (Applied Biosystems,
Warrington), with 6% denaturing polyacrylamide gels
run for 4 h at 1000 V. Allele sizes were determined
using the Genescan Analysis Version 1.2 software
package (Applied Biosystems, Warrington). Genotypes
were semiautomatically assigned using the Genotyper
software package (Applied Biosystems, Warrington).
Genotype size data were then converted into numbered
alleles and Mendelian inheritance verified using the
program GAS Version 2.0 (Dr Alan Young).
Statistical analysis
Two-point non-parametric affected sibling pair analysis was performed with the package ‘ANALYZE’
[18]. The basis of this program is the observation that
affected sibling pair analysis behaves equivalently to
LOD score analysis calculated under the assumption
of a simple recessive disease model with phaseunknown matings. Haplotype relative risk analysis was
also performed with this package.
Non-parametric multipoint analysis was performed
using the program GeneHunter Version 1.1 [19]. This
generates a non-parametric linkage score (NPL score,
which is distinct from a standard LOD score) either
from all individuals (NPL-all score) or from affected
pairs (NPL-pairs score). Compared with the NPL-all
score, the NPL-pairs score gave significantly lower P
values for TCRB, but equivalent P values for TCRA.
For simplicity, we have quoted NPL-pairs scores hereTABLE I
Microsatellite optimum PCR conditions, polymorphism information
content and number of alleles determined in pedigrees
D7S509
TCRVb6.7
D7S688
D14S50
TCRA
D14S64
MgCl
2
(mmol/l )
Annealing
temperature
(°C )
Cycles
1.0
1.5
1.5
2.0
1.5
1.5
55
56
55
55
55
56
28
35
32
28
32
35
Alleles
PIC scored
0.648
0.785
0.830
0.745
0.715
0.761
1488
1498
1388
1446
1368
1449
456
BRITISH JOURNAL OF RHEUMATOLOGY VOL. 37 NO. 4
after. Multipoint analysis utilizes genotype information
from all pedigree members to maximize the informativeness of a marker in cases of parental homogeneity
or missing genotype information. The GeneHunter
program also takes account of all pedigree information,
whereas the affected sibling pair method does not use
linkage information from distantly related affected
individuals in a pedigree. Two of the Newfoundland
pedigrees were too large and complex for analysis by
this program, and were therefore simplified by the
removal of loops (i.e. where individuals are related by
more than one pathway of descent) and unaffected
pedigree members (who contribute little linkage
information).
Exclusion mapping was performed using the program ASPEX [20]. Unlike GeneHunter, ASPEX can
only analyse nuclear families, and therefore the pedigrees were divided into component nuclear families for
this analysis. ASPEX assesses the power of a study to
exclude the presence of a susceptibility locus with
genetic effect measured in terms of the ratio lambda
[the expected sharing of 0 alleles identical by descent
(IBD) divided by the observed sharing]. Higher values
of lambda represent larger genetic effects.
RESULTS
The polymorphism information contents and
number of alleles scored for each marker are given in
Table I. Recombination fractions calculated by the
program GAS closely approximated the published
distances.
Evidence of linkage at a significance level of P < 0.05
was seen for the marker D7S688 (LOD score 1.1)
using the package ANALYZE. No other marker
achieved statistical significance by two-point analysis.
Multipoint analysis confirmed the significant result at
the TCRB locus, but the maximum point of linkage
was at the TCRVb6.7 marker rather than D7S688.
Individual P values for each marker using each program are given in Table II. The maximum NPL score
obtained was 2.1 (P = 0.015) at TCRVb6.7.
Because of the potential for genetic heterogeneity
between the inbred Newfoundland pedigrees and the
British Caucasian affected sibling pair families, the two
pedigree sets were analysed separately and together.
This had only minor effects on the overall results.
Considering only the British Caucasian families, at the
TCRA locus there was a marginal increase in P values
compared with the combined results, and a small
decrease at the TCRB locus (maximal linkage achieved
at TCRVb6.7; NPL score 2.3, P = 0.01). Considering
only the Newfoundland pedigrees, there was no evidence of linkage at the TCRB locus, whereas at the
TCRA locus there was weak evidence of linkage (maximal linkage achieved at D14S50; NPL score 1.6,
P = 0.04). The PIC values for the Newfoundland
pedigrees were significantly lower than for the UK
families for the markers TCRVb6.7 (0.69 vs 0.79) and
D14S72 (0.64 vs 0.75), but were similar for other
markers. This would reduce the power to detect linkage
at these markers in the Newfoundland pedigrees. There
was no evidence of allelic association using the haplotype relative risk test.
The exclusion analysis results are presented in Fig. 1.
This analysis assesses the strength of exclusion of the
presence of loci with different genetic effects as assessed
by the ratio lambda (the ratio of the expected and
F. 1.—Exclusion graph for TCRA using the package ASPEX. A
LOD score of less than –2.0 is generally considered to exclude
significant linkage for a susceptibility locus with genetic effect equal
to or greater than the lambda value considered. Lambda refers to
the ratio of the expected and observed sharing of 0 haplotypes
identical by descent, and is a measure of the strength of linkage of
a marker with disease.
TABLE II
Results of non-parametric analysis using the programs ANALYZE [P values and identical-by-descent (IBD) allele sharing] and GeneHunter
(P value derived from NPL-pairs statistic)
ANALYZE
D7S509
TCRVb6.7
D7S688
D14S50
TCRA
D14S64
GeneHunter
Sharing IBD (1:0)
LOD score
P
NPL score
P
133.8:110.7
168.0:138.0
158.7:123.0
135.3:128.1
118.5:115.1
135.4:114.9
0.44
0.71
1.1
0.09
0.01
0.45
0.077
0.013
0.010
0.27
0.41
0.07
1.7
2.1
1.8
0.0
− 0.1
0.3
0.043
0.015
0.031
0.51
0.54
0.38
BROWN ET AL.: TCR POLYMORPHISMS AND SUSCEPTIBILITY TO AS
observed sharing of 0 haplotypes IBD). These results
exclude the presence of a locus with genetic effects
equal to or greater than lambda = 1.6.
DISCUSSION
These findings provide weak evidence of possible
germline-coded susceptibility to AS close to or within
the TCRB locus with no evidence of an effect from
the TCRA/TCRD locus. The finding that adjacent
markers (D7S509, TCRVb6.7, D7S688) give complementary results makes it unlikely that this is an experimental error, but it could still be a false-positive
finding. It has recently been demonstrated that falsepositive linkage peaks tend to occur over shorter
genetic distances, and that peaks over a wider interval
are more likely to be true positive findings [21]. This
would support our findings being true positives.
Threshold levels of significance for reporting linkage
in genome-wide and ‘candidate’ gene scans are controversial [22]. It has been proposed that candidate gene
studies should be treated identically to genome-wide
scans, even though the number of independent loci
examined may be a small percentage of those screened
in a genome-wide scan [23]. This would correspond to
a P value of 7 × 10−4 for ‘suggestive’ linkage and
P < 2 × 10−5 for ‘significant’ linkage. As only two
candidate regions were assessed in this study, we
consider this an excessively conservative requirement.
Nonetheless, the level of significance of our findings is
not great, and further family studies will be required
for confirmation.
We have also excluded the presence of a significant
susceptibility gene in AS at the TCRA locus. Using
the program ASPEX, we have excluded the presence
of a susceptibility gene with at least moderate effects
in these families ( lambda 1.6, equivalent to the
effect of HLA in rheumatoid arthritis) [16 ]. This would
represent a much weaker susceptibility locus than HLA
in AS, for which lambda = 4.5 [8]. This may also be
a conservative estimate of the exclusion power of this
study. The program ASPEX requires that the pedigrees
analysed be nuclear. Thus, some larger pedigrees (in
particular, the Newfoundland pedigrees) had to be
broken up into smaller units for analysis by this
program, with consequent loss of power. This is not a
requirement of the GeneHunter package, which may
therefore have been more powerful in this analysis.
However, even the performance of this program is not
ideal, as it is computationally intensive and therefore
can only process families of moderately large size
(effectively in our experience ∏18 founders) [19]. This
limitation also required simplification of the largest
Newfoundland pedigrees, also with some consequent
loss of power. This study has, at best, moderate power
to detect linkage with loci causing modest small to
moderate increases in disease susceptibility. To achieve
a power of >80% to detect a locus increasing the
sibling recurrence risk (sibling recurrence/population
prevalence) by 2.0 requires 200 nuclear families assuming a fully informative marker 5 cM from the locus
concerned [24]. Although our markers were not fully
457
informative, they all had high PIC (polymorphism
information content) values (see Table I ) and the ability to determine sharing IBD was further enhanced by
the use of multipoint analysis. Further, two of our
markers were intragenic, and therefore power would
not be lost by recombination events between the gene
and the marker. Therefore, the power of this study
may be greater than that suggested by this theoretical
analysis.
No allelic association was noted between microsatellite loci and AS, but this does not formally exclude an
association between either the TCRB or TCRA/D loci
and AS. The TCR loci are recombination hot spots,
and linkage disequilibrium between biallelic polymorphisms both between and within V gene segments
is often weak [13, 25]. Microsatellites also have high
mutation rates, and therefore allelic association is more
likely to be complex (with more than one microsatellite
allele) and weaker due to loss of linkage disequilibrium
between the ancestral disease causing mutation and
alleles of the microsatellite. Therefore, it is theoretically
possible to find no microsatellite allelic association
even in the presence of a significant disease susceptibility locus such as has been observed in type 1 diabetes
at the insulin locus [26 ].
Positive associations between TCRV gene segment
polymorphisms and both rheumatoid arthritis [27, 28]
and juvenile chronic arthritis [29, 30] have been
reported, although these have not been consistent
findings. We have recently reported the absence of
linkage between the TCRA/D and TCRB loci and
rheumatoid arthritis [16 ]. To our knowledge, only one
small study of TCR gene polymorphisms in AS (49
patients, 22 controls, one C region polymorphism
assessed at each locus) has been reported, with negative
results [31]. The TCR loci are highly polymorphic,
and the exact number of single-base polymorphisms is
unknown [32]. Twenty-nine microsatellite repeats have
been identified in the TCRB gene, of which 19 are
known to be polymorphic and therefore could be used
to create a comprehensive linkage disequilibrium map
of the gene using simplex families (one patient and
their parents) to refine the region of interest [32].
Although this study provides some weak evidence
of the presence of a susceptibility locus for AS close
to or within the TCRB locus, this is far from definitive
and further studies will clearly be required to confirm
this initial observation. It is unlikely that either the
TCRA/D or TCRB loci contain genes responsible for
more than a proportion of the non-MHC genetic
susceptibility in AS.
A
Grant support for this project was received from the
Arthritis and Rheumatism Council ( UK ), the John
Coates Foundation Trust, the R. J. Harris Charitable
Trust, the Col. W. W. Pilkington Charitable Trust,
and the Rehabilitation and Medical Research Trust.
CIA receives support from grant AR44422 ( US
National Institutes of Health, the National Institute
for Arthritis, Musculoskeletal and Skin Diseases).
458
BRITISH JOURNAL OF RHEUMATOLOGY VOL. 37 NO. 4
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