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Supplementary Material
Figure S1. Haplotype dependent differences in the expression of the membrane bound (H20)
and soluble (H6) IL-7RA isoform. Results are presented as means ± s.d.; ***P ≤ 0.001.
Figure S2. Haplotype differences in IL-7RA surface expression. PBMCs of MS patients
homozygous for either haplotype 1 or haplotype 3 (number of samples between 13
and 18) were specifically stained for CD4+, CD4+ naïve, CD4+ memory, CD8+, CD8+
naïve, CD8+ memory, CD56dim and DC sub-populations (CD1c, CD141, CD303) and
analyzed by flow cytometry. Results are presented as means ± s.d.
Figure S3. (A) Multidimensional scaling of gene expression values of unstimulated CD4+ T
cells. A subset (843 unique Ensembl gene IDs) corresponding to genes with low intragroup variability (lower third of median absolute deviation per haplotype as threshold)
was used. Color coding: haplotype 1, haplotype 2, and haplotype 3 are plotted as
black, green, and red spheres, respectively. (B) Principle component analysis of CD4 +
T cells before and after stimulation with IL-7. Genes that respond to IL-7 with
uncorrected p-value ≤0.05 (7036 unique Ensembl gene IDs) were used.
Figure S4: Results from nonnegative matrix factorization. (A) Consensus clustering using
four algorithms (nsnmf, offset, lee, brunet, see (Gaujoux, R. & Seoighe, C. A flexible R
package for nonnegative matrix factorization. BMC Bioinformatics 11, 367 (2010)) for
details) showing cophenetic coefficients for a range of factorization ranks. A pilot
analysis on the data set was done with 50 runs with each algorithm. (B) Residuals
reported by the NMF process for four algorithms (nsnmf, offset, lee, brunet). Results
from a pilot analysis on the data set with 50 runs with each algorithm are shown. (C)
NMF consensus matrix showing congruent placing of individuals into a given class.
The correlation matrix was obtained from the run that achieved the lowest
approximation error across 1000 runs with random seeding using the non-smooth
nonnegative matrix factorization algorithm. (D) Heatmap of the mixture coefficients (H).
Contribution of each metagene to explain the expression profile for individual samples
the input data matrix is shown.
Table S1. Gene expression in CD4+ T cells of MS individuals homozygous for either Hap1,
Hap2, or Hap3. Transcript levels from genes varying maximally between haplotypes
(top 1% divergent gene expression from F-test statistics, p-value ≤ 0.02) are listed.
Columns hold data for: Ensembl ID; Symbol; p-value, p-value from ANOVA F-test
giving the significance that any comparison differs from the null hypothesis that there
are no differences in expression levels between haplotypes; logFC, reports the
differences between the respective haplotype averaged expression levels (i.e., logFC
Hap1 vs Hap3 displays data for Hap1 versus Hap3); for clarity reasons, negative
values are plotted in red.
Table S2. Genes induced by stimulating isolated CD4+ T cells with IL-7. Top 1% of
significantly regulated genes corresponding to adjusted p-value ≤ 1.6 E-5. Expression
differences were calculated by combining samples according to stimulus (either
unstimulated or stimulated) irrespectively of their haplotype assignment. Columns
display data for: Ensembl ID; Symbol; adj. p-value, multiple test-adjusted p-value
(false discovery rate = 0.05) for expression differences between unstimulated and
stimulated conditions; logFC, as explain above for Supplementary Table S1.
Table S3. Genes responding to IL-7 but showing haplotype-associated differences in
response. Genes were selected based on IL-7 responses (adjusted p-value ≤ 0.05 in
at least one of three haplotype groups) in combination with differences in response
between haplotypes (F test from three-group test on expression changes induced by
IL-7, p-value ≤ 0.05, 209 genes; see also Supplementary Table S1 for explanation).
For each gene the results from NMF (see methods) are given as contribution
(percentage) to one of the three metagenes derived by the method. Top metagene
column indicates that this gene is among the top 10% most relevant metagene
describing features. Columns hold data for: Ensembl ID; Symbol; LogFC reports the
differences between haplotype averaged expression levels (stimulated versus
unstimulated condition); adj. p-value, multiple test-adjusted p-value (false discovery
rate = 0.05) for expression differences between unstimulated and stimulated
conditions (both logFC and adj. p-values are given separately for each haplotype);
HapX vs HapY difference, logFC difference in response to IL-7 (see analogous
columns for unstimulated condition in Supplementary Table S1); p-value from ANOVA
F-test (as explained above); MetageneX, contribution of gene (in percent) to the
characteristics of the given metagene; Top metagene, indicates that this gene is
among the top 10% most relevant metagene describing features.