Association between reduced copy-number at T-cell receptor gamma
and childhood allergic asthma: a possible role for somatic mosaicism
Kyle Walsh, Michael Bracken, William Murk, Josephine Hoh, Andrew DeWan
Center for Perinatal Pediatric and Environmental Epidemiology, Yale School of Public Health. Department of Epidemiology and Public Health, Yale University
Introduction
Somatic copy-number at TCRα among PRAM subjects
6
5
Copy Number
Asthma is a chronic inflammatory disease of the lungs which affects more than 6.5 million
American children. A family-based genome-wide association study of copy-number variation
identified an association between decreased copy-number at TCRγ and childhood allergic
asthma1. TCRγ encodes the T-cell receptor gamma glycoprotein, a cell-surface protein found on
T-cells and involved in cell-mediated immunity. Copy-number variation at these loci is wellknown, but appears to be dominated by somatic mutations, as opposed to germline events1,2.
Using quantitative real-time PCR, we sought to determine if copy-number variation at TCRα,
TCRβ or TCRγ was associated with childhood allergic asthma in an independent cohort of 94
cases and 455 controls using DNA from buccal swabs.
Table 1: Statistical analyses of copy-number genotypinga results at each of three TCR genes
Points above
pooled median
45.90
220.10
NA
Expected Mann-Whitney Median Test
p-valueb
p-valuec
under H0
45.50
p = 0.7972
p = 0.9343
220.50
NA
TCRα
Case
Control
Pooled
th
th
N Median 10 %ile 90 %ile
91
1.98
1.59
2.36
441
1.98
1.52
2.35
532
1.98
1.53
2.35
TCRβ
Case
Control
Pooled
90
437
527
2.01
1.98
2.00
1.49
1.43
1.44
2.49
2.45
2.47
47.00
216.00
NA
44.90
218.10
NA
Case
TCRγ1 Control
Pooled
94
454
548
1.95
1.99
1.98
1.81
1.80
1.80
2.19
2.26
2.26
34.57
239.43
NA
47.00
227.00
NA
6
Case
TCRγ2 Control
Pooled
91
435
526
1.93
1.99
1.98
1.63
1.66
1.65
2.16
2.25
2.25
39.75
223.25
NA
45.50
217.50
NA
5
aSomatic
4
Controls
3
Cases
2
p = 0.8585
p = 0.6284
p = 0.0199
p = 0.0044
p = 0.0488
p = 0.1805
1
Methods
0
0
100
200
300
400
500
600
Patient ID
Statistical Analysis
The primary statistical test utilized to compare copy-number values was the Mann-Whitney U
test. Statistical analyses were performed using SAS version 9.1 (SAS Institute; Cary, NC). All pvalues reported are two-sided.
Tests of Mendelian Inheritance
To determine if the CNVs identified in this study are inherited, or if they are somatic mutations, a
subset of individuals determined to have CNVs at TCR loci and the parents of these individuals
were genotyped (n = 27 trios). Lack of Mendelian consistency in CNV inheritance was
considered indicative of somatic mutations, as germline CNVs are known to be stably inherited
and de novo germline events are quite rare3.
Results
Copy Number
4
Controls
3
Cases
2
copy-number was determined using real-time quantitative PCR. CT values for the target and reference assay
were imported into CopyCaller™ Software version 1.0 (Applied Biosystems; Foster City, CA) and analyzed using
comparative CT (ΔΔCT) relative quantification. These continuous copy-number values can be interpreted as the
average copy-number within a mosaic cell population. Two different primer sets were used to genotype TCRγ,
targeting positions separated by 1730 bp.
bThe Mann-Whitney test (a.k.a. Wilcoxon rank-sum test) ranks copy-number values and tests whether those among
cases are equally distributed compared to those among controls.
cThe Median test assesses whether the median copy-number value among cases is equal to the median copy-number
value among controls.
1
0
0
100
200
300
400
500
Table 2: Parent-child trio genotypesa
600
Child's genotype Number of trios genotyped
TCRα deletion
5
TCRα duplication
6
TCRβ deletion
4
TCRβ duplication
5
TCRγ deletionb
0
TCRγ duplication
7
Patient ID
Somatic copy-number at TCRγ1 among PRAM subjects
6
5
Trios showing Mendelian inheritance
1
3
2
1
NA
1
aFor
4
Controls
3
Cases
2
1
0
100
200
300
400
500
600
Patient ID
Figure 1. Distribution of individuals’ average somatic copy-number in
mosaic buccal cell populations at each of three TCR genes (N = 94 cases,
454 controls). Copy-number did not differ significantly by case/control
status at TCRα or TCRβ (p = 0.7972 and 0.8585, respectively). Cases had
significantly lower copy number values at TCRγ1 (p = 0.0199), as
demonstrated by the larger proportion of case values (versus control
values) falling below the copy-neutral value of 2.0 in the figure.
Human Chromosome 7p14.1
Cen
38.39Mb
38.25Mb
38.285Mb
Hs03646230
38.283Mb
Hs04326912
a copy-neutral child, the only parental genotype which is inconsistent with
Mendelian inheritance is a homozygous deletion. Therefore, parents of children
determined to be copy-neutral were not genotyped because such trios are overwhelmingly
uninformative for testing Mendelian inheritance of CNVs.
bNo children with TCRγ deletions had paternal DNA available. TCRγ copy-number
genotypes presented here are those determined using the first TCRγ probe (ABI primer ID
Hs04326912_cn).
Discussion
0
Tel
Genotyping results indicated that copy-number variants at these genes are largely somatic
mutations, as inheritance did not show Mendelian consistency. In these mosaic cell populations,
copy-number was significantly reduced among asthmatic children at both TCRγ probes (p =
0.0199 and 0.0488), but was not associated at TCRα or TCRβ (p = 0.7972 and 0.8585,
respectively). These findings support the association between reduced copy-number at TCRγ and
childhood allergic asthma. Our study utilized DNA derived from buccal cells and demonstrates
that these somatic events are not limited to blood-derived DNA. As these are somatic mutations,
our results can not determine whether reduced TCRγ copy-number is a risk factor for allergic
asthma, or is an immunologic response to the disease.
Somatic copy-number at TCRβ among PRAM subjects
Copy Number
Copy-number Genotyping
Copy-number genotyping was performed using real-time quantitative PCR and commercially
available reagents (Applied Biosystems) following the manufacturer’s recommendations. Cases
and controls were assigned to one of five plates for genotyping using 1:5 blocked randomization.
A copy-number reference assay containing two primers and a VIC and TAMRA dye-labeled
probe assayed copy-number at the RNaseP locus. This was amplified in a multiplex reaction with
a copy-number target assay containing two primers and a FAM dye-labeled MGB probe which
targeted a region in one of the TCR genes. Plates were run on the ABI 7900HT machine using
the manufacturer’s recommended PCR cycling conditions.
Cycle thresholds were calculated using the SDS v2.2.2 software. Wells with a cycle threshold
exceeding 32.5 for either the target or the reference probe were excluded from analysis. 549 of
574 samples had 2 or more replicates successfully amplify in at least one TCR gene assay and
were included in the final analysis. This included 94 cases and 455 controls.
CT values for the target and reference assay were imported into CopyCaller™ Software version
1.0 (Applied Biosystems; Foster City, CA). A comparative CT (ΔΔCT) relative quantification
analysis of the real-time data was performed. First, the difference between the target and
reference CT value is determined for each well and then averaged across sample replicates. The
ΔCT values of the samples are used to calculate copy-number at the target locus relative to that at
the RNaseP control locus, which is known to exist at one copy per haploid genome. Continuous
copy-number values represent the average somatic copy-number in a mosaic cell population, and
those in cases were compared to those among the controls.
Figure 2. Graphical representation of the location of qPCR probes in the
TCRγ gene. Solid blue bars represent previously reported copy-number
variable regions.
Our results support the association between childhood allergic asthma and reduced copy-number
at TCRγ. Further work is needed to resolve whether these mutations predispose individuals to
asthma, or whether CNVs at this position arise as a result of the disease. If they do arise as a
result of the disease, it will be valuable to determine if this is a coincidental marker of the asthma
phenotype, or if deletion of this gene is an immunologic response which establishes a cell
population that is mosaic for TCRγ copy-number.
Reduced TCRγ copy-number is expected to correlate with a reduction in the proportion of total Tcells expressing a γδ TCR, as opposed to an αβ TCR. As these T-cell subpopulations vary in their
inflammatory effects, their involvement in the etiology of allergic asthma seems plausible.
References
1. Ionita-Laza, I., Perry, G.H., Raby, B.A., et al. (2008) On the analysis of copy-number
variations in genome-wide association studies: a translation of the family-based association test.
Genet Epidemiol, 32, 273-84.
2. Diskin, S.J., Hou, C., Glessner, J.T., et al. (2009) Copy number variation at 1q21.1 associated
with neuroblastoma. Nature, 459, 987-91..
3. Sebat, J., Lakshmi, B., Malhotra, D., et al. (2007) Strong association of de novo copy number
mutations with autism. Science, 316, 445-9.