1 Supplementary Figure 1 STAT3 expression in HEK293 cells Western blot showing the expression of STAT3 protein in HEK293 cells transfected with STAT3 constructs. HEK293 cells were lysed, and protein extracts were probed with anti-STAT3 antibody. β-actin was used as a loading control. The experiment was repeated twice with similar results. Nature Genetics: doi:10.1038/ng.3040
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Supplementary Figure 1
STAT3 expression in HEK293 cells
Western blot showing the expression of STAT3 protein in HEK293 cells transfected with STAT3 constructs. HEK293 cells were lysed,and protein extracts were probed with anti-STAT3 antibody. β-actin was used as a loading control. The experiment was repeated twicewith similar results.
Nature Genetics: doi:10.1038/ng.3040
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Supplementary Figure 2
Genotype-phenotype relationship in STAT3 alterations.
The predicted effects of the STAT3 alterations were modeled in PDB structure 1BG1 (mouse STAT3/DNA complex) using SWISS-MODEL and visualized in the Swiss-PdbViewer. (a) Overview of the STAT3 dimer bound to DNA; STAT3 chains are shown in ribbonform, with residues N646 (red) and N647 (green) shown as space-filling residues on the left chain only; DNA strands are shown as blue and turquoise ribbons. (b) As in a, but expanded to show the proximity of residues N646 and N647 to both the DNA-binding and dimerization surfaces. (c) Predicted molecular surfaces of wild-type STAT3 (wt) and mutants N646K, N647D and N647I; surfaces are colored for positive charge (blue; top row), negative charge (red; middle row) and hydrophobicity (brown (most polar) to blue (most hydrophobic); bottom row); structures have been rotated compared to in a and b to show relevant groups more clearly. The N646K alteration reported here results in increased positive charge (circled, N646K column, upper row) at the DNA-binding surface; this is likely to result in higher DNA binding affinity due to electrostatic interaction with the DNA backbone and, hence, increased STAT3 activity. Conversely, the N647D substitution, previously reported as a loss-of-function alteration in HIES, leads to increased negative surface charge in this region (circled, N647D column, middle row) and is likely to inhibit DNA binding and/or dimerization. By
Nature Genetics: doi:10.1038/ng.3040
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comparison, a different substitution at this position, N647I, has been previously reported as an activating alteration in LGLL; it has been postulated that STAT3 mutations in LGLL promote STAT3 dimerization and, hence, biological activity, as a result of increased hydrophobicity at the dimerization surface. This is consistent with protein modeling in silico, which predicts increased hydrophobicity in this region (circled, N646I column, bottom row) compared to wild-type STAT3 or other variants.
Nature Genetics: doi:10.1038/ng.3040
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Nature Genetics: doi:10.1038/ng.3040
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Supplementary Figure 3
Increased basal STAT3 activity in vitro.
The intracellular expression of IFN-γ and TNF-α was measured from unstimulated and stimulated (anti-CD3, anti-CD28, anti-CD49d) CD4+ and CD8+ T cells after 6-h incubation using flow cytometry. Samples were available from six healthy controls and two patients (patient 3 (p.K392R) and patient 5 (p.K658N)). In all analyzed cases, the expression of IFN-/TNF- was under 1% in unstimulatedcells. The median percentage of stimulated cytokine-producing CD4+ and CD8+ cells was 8.0% and 12%, respectively, among healthy controls (data of one control shown). CD4+ cells from patient 2 showed increased cytokine production when stimulated. IFN, interferon;TCR, T cell receptor; TNF, tumor necrosis factor.
Nature Genetics: doi:10.1038/ng.3040
Supplementary Table 1. Breakdown of variants identified by exome sequencing of individual 1
Individual 1 substitutions indels
Total passing quality filters 18220 570
After dbSNP131 filtering 466 489
After 1000Genomes filtering 95 16
After excluding those in parents (de novo) 18 1
After excluding those outside of the coding regions and conserved splice sites (+/- 2bp) 15 0
After excluding non-synonymous and intronic variants 7 0
After manual inspection and exclusion of putative de novo variants present in a parent 1 0
Nature Genetics: doi:10.1038/ng.3040
Supplementary Table 2: Features of 25 individuals with multiple early onset-autoimmune disease
sequenced for STAT3 mutations
Pat
ien
t
Earl
y-o
nse
t d
iab
ete
s
Au
toim
mu
ne
Ente
rop
ath
y
Au
toim
mu
ne
pu
lmo
nar
y d
isea
se
A
uto
imm
un
e th
yro
id d
ysfu
nct
ion
Au
toim
mu
ne
join
t d
isea
se
De
nta
l an
om
alie
s
STAT3
mu
tati
on
iden
tifi
ed
1 p.T716M
2 p.K392R
3 p.N646K
4 p.K658N
5 No
6 No
7 No
8 No
9 No
10 No
11 No
12 No
13 No
14 No
15 No
16 No
17 No
18 No
19 No
20 No
21 No
22 No
23 No
24 No
25 No
Nature Genetics: doi:10.1038/ng.3040
Supplementary Table 3: Clinical characteristics of individuals with a de novo STAT3 mutation
1 Otonkoski, T. et al. Diabetologia 43, 1235-1238,(2000). 2 Sjoroos, M. et al BioTechniques 18, 870-877 (1995). 3 Gambineri, E. & Torgerson, T. R. et al CMLS 69, 49-58, (2012). 4 Peterson, P. & Peltonen, L.. Journal of autoimmunity 25 Suppl, 49-55,(2005). 5 d'Hennezel, E. et al Journal of medical genetics 49, 291-302, (2012). 6 Wildin, R. S. et al Journal of medical genetics 39, 537-545 (2002). 7 Bezrodnik, L. et al Clinical and experimental immunology (2013). 8 Lohr, N. J. et al. American journal of human genetics 86, 447-453 (2010). 9 Koskela, H. L. et al. N Engl J Med 366, 1905-1913 (2012)