Supplementary Information
Early-onset Multi-Organ Autoimmunity Caused by Activating Germline Mutations in
STAT3
Sarah E. Flanagan,1, 13 Emma Haapaniemi,2,13 Mark A. Russell,1,13 Richard Caswell,1 Hana Lango Allen,1
Elisa De Franco,1 Timothy J. McDonald,1 Hanna Rajala,3 Anita Ramelius,4 John Barton,5 Kaarina
Heiskanen,6 Tarja Heiskanen-Kosma,7Merja Kajosaari,8 Nuala P. Murphy,9 Tatjana Milenkovic,10 Mikko
Seppänen,11 Åke Lernmark,4 Timo Otonkoski,6 Satu Mustjoki,3 Juha Kere,2,12 Noel G. Morgan,1 Sian
Ellard1 and Andrew T. Hattersley1
1
Institute of Biomedical and Clinical Science, University of Exeter Medical School, Exeter, EX2 5DW, UK
Folkhälsan Institute of Genetics, and Research Programs Unit, Molecular Neurology, University of Helsinki, Helsinki,
Finland
3
Hematology Research Unit Helsinki, Department of Hematology, University of Helsinki and Helsinki University Central
Hospital Cancer Center, Helsinki, Finland
4
Department of Clinical Sciences, Lund University/CRC, Skåne University Hospital SUS, Malmö, Sweden
5
Bristol Royal Hospital for Children, Upper Maudlin Street, Bristol, BS2 8BJ, UK
6
Children’s Hospital, Helsinki University Central Hospital and Research Programs Unit, Molecular Neurology, University of
Helsinki, Helsinki, Finland
7
Department of Pediatrics, Kuopio University Hospital, Helsinki, Finland
8
Children’s Hospital, Helsinki University Central Hospital, Helsinki, Finland
9
Department of Diabetes and Endocrinology, Children's University Hospital, Temple St., Dublin 1, Ireland
10
Department of Endocrinology, Institute for Mother and Child Health Care of Serbia ‘Dr Vukan Cupic’, Belgrade, Serbia
11
Immunodeficiency Unit, Division of Infectious Diseases, Helsinki University Central Hospital, Helsinki, Finland
12
Department of Biosciences and Nutrition, and Center for Innovative Medicine, Karolinska Institutet, Hälsovägen 7, 14183
Huddinge, Sweden
13
These authors contributed equally to this work
2
Table of Contents
Supplementary Methods …………….
Supplementary Table 1 ……………….
Supplementary Table 2 ……………..
Supplementary Table 3 ……………..
Supplementary Table 4 ……………..
Supplementary Table 5 ……………..
Supplementary Table 6 ……………..
References ……………
Supplementary Methods
Cohort selection and sample preparation
Twenty-five individuals with early-onset polyautoimmune disease (diagnosed before 5 years of age)
and 39 subjects with isolated permanent diabetes diagnosed before 6 months were recruited by their
clinicians for molecular genetic analysis in the Exeter Molecular Genetics Laboratory (n=63) or the
Folkhälsan Institute of Genetics, University of Helsinki (n=1). Genomic DNA was extracted from
peripheral leukocytes using standard procedures. All subjects and/or their parents gave informed
consent for genetic testing and institutional review board approval was received for this study.
Exome sequencing and variant calling
Genomic regions corresponding to NCBI Consensus Coding Sequence (CCDS) database were captured
and amplified using Agilent’s SureSelect Human All Exon Kit (v1). Paired-end sequencing was
performed on an Illumina GAII, one lane per sample, 101 or 76bp read length. The resulting reads were
aligned to the hg19 reference genome with BWA providing mean target coverage of 66.3 reads per
base. At least 72% of the targeted bases were covered by at least 20 reads. Variants were called with
GATK UnifiedGenotyper and annotated using Annovar and SeattleSeq Annotation server, as previously
described.1 Variant filtering steps are shown in Supplementary table 1.
STAT3 sequencing and microsatellite analysis
Sanger sequencing was undertaken in patient 1 and her unaffected parents to confirm that the
p.T716M STAT3 variant had arisen de novo. Exons 2-24 and intron/exon boundaries of STAT3
(NM_139276.2) were Sanger sequenced in a further 24 individuals with at least 2 early-onset
autoimmune features of unknown cause (Supplementary table 2). Primers for STAT3 exons 2-24 are
provided in Supplementary table 3.
Targeted next-generation sequencing of STAT3 was undertaken on a further 39 individuals with
isolated permanent diabetes diagnosed before the age of 6 months of unknown cause without
additional autoimmune features. All patients were less than 5 years of age at the time of genetic
testing. We adapted our custom Agilent SureSelect exon-capture assay (Agilent Technologies, Santa
Clara, CA, USA) to include baits for exons 2-24 and intron/exon boundaries of STAT3 (sequences
available on request to authors.2 Samples were fragmented using a Bioruptor (Diagenode, Liège,
Belgium), indexed for multiplexing and hybridised (in pools of 12 samples) according to the
manufacturer’s instructions. Sequencing was performed with an Illumina HiSeq 2000 (Illumina, San
Diego, CA, USA) (48 samples per lane) and 100 bp paired end reads. Data were processed to identify
potential pathogenic mutations located within 50 bp upstream and 10bp downstream of each exon.
We identified STAT3 mutations in a further 3 individuals with early onset autoimmune disease (4 of 25,
16% of cohort) and 1 individual with permanent neonatal diabetes (1 of 39, 2.6% of cohort). This
brought the total number of STAT3 positive subjects to 5. Sanger sequencing of parental samples
confirmed all mutations had arisen de novo. Biological relationships were confirmed by microsatellite
analysis using the PowerPlex kit (PowerPlex 16 System, Promega, Southampton, UK).
In total four different mutations were identified in 5 unrelated individuals. All mutations affected
residues in the highly conserved DNA binding domain (x1), SH2 domain (x2) and transactivation domain
(x1) (conserved to Zebrafish). None of the mutations were present in dbSNP132, 1000 Genomes
Project database (based on 1094 individuals) or the Exome Sequencing Project (based on 6500
individuals).
Functional studies of STAT3 mutations
Mutations within human STAT3 (Source Bioscience) were generated using the QuikChange sitedirected mutagenesis kit following the manufacturer’s guidelines (Agilent Technology). The primer
pairs used to generate each mutant are provided in Supplementary table 4. The success of all
mutatagenesis reactions was confirmed by direct sequencing of the entire STAT3 insert (Eurofins).
Following mutagenesis, STAT3 inserts were subcloned into the multiple cloning site of a
pcDNA5/FRT/TO expression vector between AflII and EcoRV restriction sites.
The transcriptional activity of STAT3 was assessed via a STAT3 responsive dual firefly/Renilla luciferase
Cignal reporter system (Qiagen). HEK293 cells were seeded at a density of 1 x 10 5 cells/well, and were
transfected after 24h with a combination of 200ng Cignal reporter assay constructs and 400ng WT or
mutant STAT3 pcDNA5/FRT/TO using the Attractene transfection reagent according to the
manufacturer’s instructions (Qiagen). Cells were incubated in the transfection mix for 24h and, where
appropriate, 20ng/ml IL-6 was also included for the final 18h. STAT3 reporter activity was assessed
using a dual luciferase reporter assay system (Promega). To confirm that equivalent amounts of STAT3
protein were expressed following transfection of each construct, cells were lysed and protein extracted
prior to Western blotting with anti-STAT3 antibody (Cell Signalling) as described previously5.
FACS analysis of patient cells
For the assessment of T-cell activation and degranulation, fresh mononuclear cells from patients 2 and
5 (supplementary table 5) were stimulated for 6 h with anti-CD3, anti-CD28 and anti-CD49d (BD
Biosciences). The cells were analyzed using 4- or 6 colour flow cytometry panel with mAbs against
the antigens CD3, CD4, CD8, IFN-γ and TNF-α.
Supplementary Table 1. Breakdown of variants identified by exome sequencing of individual 1
Individual 1
Total passing quality filters
After dbSNP131 filtering
After 1000Genomes filtering
After excluding those in parents (de novo)
After excluding those outside of the coding
regions and conserved splice sites (+/- 2bp)
After excluding non-synonymous and intronic
variants
After manual inspection and exclusion of
putative de novo variants present in a parent
substitutions indels
18220
570
466
489
95
16
18
1
15
0
7
0
1
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
















































STAT3 mutation identified
Dental anomalies
Autoimmune joint disease
Autoimmune thyroid dysfunction









Autoimmune pulmonary disease
Autoimmune Enteropathy
Early-onset diabetes
Patient
Supplementary Table 2: Features of 25 individuals with multiple early onset-autoimmune disease
sequenced for STAT3 mutations
p.T716M
p.K392R
p.N646K
p.K658N
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
Supplementary Table 3: STAT3 primers for Sanger sequencing
Exon
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18F
18R
19
20
21
22
23
24
Forward Primer Sequences (M13 tailed)
Reverse Primer Sequences (M13 tailed)
All primers start 5’ TGTAAAACGACGGCCAGT
All primers start 5’ CAGGAAACAGCTATGACC
CCCCAGAGCATCTTTATCCC
GGGTTATAGCATCAGGTTTGC
TAGTAACGACCTCCCCTTCG
TTCCCTTCCTCTTGTGATGG
AACAGGGAGCCTTCTCTTGG
GGAGGTACGGGTCCTCAAAG
ATTTCAGCGTCTTGTGGCAG
TTTTCAGCATCCACCCAAC
GGTAATTTAGCATCCTTGTCCC
GACAGCTTGGCCTATTTACCTG
TGCGCTGATCAACTGTAACTG
ATTCCCACATCTCTGCTCCC
ATGGAAGAATCCAACAACGG
TGCTGCTTAGACTGGTCTCG
CACTCCTCGCCTAGAGTTGG
AACATGCTGACCAACAATCC
AAATCCTCAGGCCCGTCTAC
CCTTCAAAGATGTGAAAGCTG
CTGAACTCTTGGTCCAGCG
GCTGGCAAGGGCTTCTC
CACTACAATTCTTTCCCATAAGGAG
TAAATGAGGGCAGACAACCC
AGCCCCTGGGCTATGTTTAG
TCCAGGGAGGAGGGTAAATC
CTCATTTTCCCCATCACCTG
AAGTATACAGAGCTTTGAGAAAGGG
TCTGTTGGATTCTTTTGGTGG
CAAGAGAAGGCTCCCTGTTG
ATGACCAGGCTCCTTTGAGG
CAACTCCAGAGCAGGAACTTCT
GCTAAATTTGAATATGGAAAAGTCC
GGAAAGAGAAGATGGGCTCAC
ATGGCAACAAATTTCAACCC
TGTCCACAAAATGAAGATCTCTG
ATTCCCACATCTCTGCTCCC
TGCGCTGATCAACTGTAACTG
GTTCATGTCACTTTGGCCTG
CCCCTGTACGTAGCCTCTCA
GTCCTCGCTTGGTGGTG
GCCTTGCTCAGGAAAGAAAC
CCTTCAAAGATGTGAAAGCTG
AAATCCTCAGGCCCGTCTAC
AAAGCCCATGATGTACCTGG
AAGCAAACCAATCCTTCAGC
AACAGGGTGTTCAGGGTCTC
TCAAACTCTGGTCTCCAACAG
TCTCTTTTGGAAAGCAAAGCTC
AGCAGATCACCCACATTCAC
Supplementary Table 4: Primer sequences for site-directed mutagenesis
Mutation
Forward Primer
Reverse Primer
Polyautoimmune mutations:
p.K392R
ATTCTGGGCACAAACACAAGAGTGATGAACATGGAAG
TCTTCCATGTTCATCACTCTTGTGTTTGTGCCCAGAATG
p.N646K
ACACAAAGCAGCAGCTGAAGAACATGTCATTTGCTG
AGCAAATGACATGTTCTTCAGCTGCTGCTTTGTG
p.K658N
TCATCATGGGCTATAACATCATGGATGCTACC
TGGTAGCATCCATGATGTTATAGCCCATGATG
p.T716M
ATCTGTGTGACACCAATGACCTGCAGCAATACC
TGGTATTGCTGCAGGTCATTGGTGTCACACAG
Hyper IgE mutations:
p.R382W
AGCTCTCAGAGGATCCTGGAAATTTAACATTCTGGGC
TGCCCAGAATGTTAAATTTCCAGGATCCTCTGAGAGC
p.V637M
AGACCCAGATCCAGTCCATGGAACCATACACAAAG
TGCTTTGTGTATGGTTCCATGGACTGGATCTGGGTC
Supplementary Table 5: Clinical characteristics of individuals with a de novo STAT3 mutation
PATIENT 1
PATIENT 2*
PATIENT 3
PATIENT 4
PATIENT 5
Table of clinical
features
Mutation
Sex
Current Age (yrs)
Birth weight (SDS)
p.Thr716Met
(c.2147C>T)
Female
6
-1.59
p.Lys392Arg
(c.1175A>G)
Female
15
-5.81
-6.63
Delayed puberty
p.Asn646Lys
(c.1938C>G)
Male
6
-2.70
p.Asn646Lys
(c.1938C>G)
Male
3
-1.59
-1.47
-2.05
0
40.3 RU (<5.36)
0.08 (<0.43)
Not tested
3224 RU(<1.56)
3
5 (<34)
0(<5)
11(<60)
Not tested
insulin treated
Negative
43
31.9 U/mL (<1.0)
9.5 U/mL (<5)
Not tested
Not tested
insulin treated
Not tested
Not diabetic
Negative
Negative
Not tested
Negative
Fecal elastase
166mg/ g faeces (>200)
On enzyme
replacement therapyLow Fecal elastase
NO
NO
NO
High Risk
DRB1*03 -DQB1*02/
DRB1*03 -DQB1*02
High Risk
DRB1*04 DQB1*0302
Low Risk
DQA1*01DQB1*05/
DQA1*01DQB1*0602
Low Risk
DQA1*03B1*03:02/A1*01B1*06:02
Low risk
1.8
1.5
0.76
0.8
N/A
61
70
68
69
Not tested
Coeliac disease
Coeliac-disease
NO
NO
Autoimmune
enteropathy
17 months
<12 months
N/A
N/A
<12 months
High Risk
DR52-DR3-DQ2
High risk
DQ8
Not tested
Not tested
Low risk
TtG IgA 20.47 U/mL (<10)
TtG IgG > 200 U/mL (<10)
Not tested
<1.0 (<10)
2.8 (<7)
Not tested
Not tested
Positive Endomysial
& gliadin
autoantibodies
Not tested
Not tested
Positive for gliadin
autoantibodies
YES
YES
N/A
N/A
NO
NO
Mild atopic
YES
YES
Non-specific
dematitis
None
Desquamative
interstitial
pneumonitis
None
None
Asthma
Recurrent URTI
Growth (Height SDS)
-2.33
DIABETES insulin doses HbA1c
Age at Diagnosis (Wks)
2
GAD65 autoantibodies
35 RU (<34)
I-A2 autoantibodies
0 (<5)
ZnTn8 autoantibodies
7 (<60)
Not tested insulin
IAA autoantibodies
treated
ICA
Negative
Pancreatic Exocrine
Insufficiency
T1D HLA Type†
Current Insulin dose
(U/Kg/day)
Current HbA1c
(mmols/mol)
ENTEROPATHY
Type
Onset of symptoms
(months)
Coeliac HLA type
Anti TtG autoantibodies
Endomysial/gliadin
autoantibodies
Responsive to gluten
free diet
OTHER AUTOIMMUNE FEATURES
Eczema
Pulmonary
manifestations
Primary
hypothyroidism (TPO
Other features
antibodies 224 U/mL
(<20). Lipoatrophy at
sites of insulin injection
HEMATOLOGICAL FEATURES
NO
28 JDFU(<2.5)
p.Lys658Asn
(c.1974G>C)
Female
17
-1
-4.00
Delayed puberty
Negative
None
None
Juvenile arthritis,
severe dry eyes
Progressive
macular edema &
vision loss.
Kawasaki diseaselike episodes
T-cell LGL leukemia.
Hb 10.0g/dl nd
NO
Lymphadenopathy
Autoimmune
cytopenias.
MCV 64.7fl (73-89)
MCH 20.4pg (2430)
& splenomegaly.
Autoimmune
cytopenias
NO
Dentures
NO
NO
<2.0 (<100kU/L)
-
1.3% (<6%)
-
<2.0 (<100kU/L)
0.30 x109 /L
(<0.7x109/L)
<2.0 (<100kU/L)
0.28 x109 /L
(0.08-1.1x109/L)
DENTAL ANOMALIES
Delayed eruption
of primary teeth
IMMUNODEFICIENCY
Ig E
Eaosinophils
Infection susceptibility
0.7 (3.9-10)
-
Recurrent bacterial
Recurrent
LRTI, IVIG from 12
NO
NO
bacterial URTI,
y/o
IVIG from 12 y/o
Table: TPO = Thyroid Peroxidase, TtG = Anti-Tissue trans glutaminase antibody, URTI=Upper Respiratory Tract Infections, LRTI=Lower
Respiratory Tract Infections, IVIG = Intravenous Immunoglobulin Replacement Therapy. N/A = not applicable. Normal ranges are
provided in parenthesis when appropriate. * Patient previously reported in 3. †HLA typing was undertaken on patients 1-4 using
previously described methods4.
NO
Supplementary Table 6: Adapted from Gambineri and Torgerson (2012)6 using additional published literature. Approximate % of
patients affected with a condition are shown in brackets. Abbreviations APS1 (autoimmune polyendocrinopathy syndrome type I) IPEX
(X-linked immunodysregulation, polyendocrinopathy, and enteropathy)
Disorder
Gene
Inheritance
Number of
patients reported
References
STAT3
polyautoimmunity
STAT3
Dominant
5 patients
(5 families)
This paper
Common clinical
features (present
in >50% of
patients
Type 1 diabetes
(80%)
Enteropathy (60%)
Short stature
(100%)
Additional clinical
features
Autoimmunoe
thyroid (40%)
Juvenile Chromic
Arthritis (20%)
Fibrotic lung
disease (20%)
Other laboratory
or diagnostic
features
Normal eosinophil
count
Low-normal IgE
APS1
IPEX
CD25 deficiency
ITCH deficiency
AIRE
Recessive
>150
FOXP3
Recessive
>70
IL2RA
Recessive
4 patients
(4 families)
ITCH
Recessive
10 patients
(1 family)
Peterson, & Peltonen
(2005)7
d'Hennezel, et al
(2012) 8
Wildin et al(2002)9
Enteropathy (100%)
Eczematous
Dermatitis (70%)
Type 1 diabetes
(67%)
Bezrodnik, et al (2013)10
Lohr et al. (2010)11
Enteropathy (100%)
Recurrent persistent
viral infection(100%)
Eczema (75%)
Developmental delay (100%)
Macrocephaly(90%)
Dysmorphic facies (90%)
Chronic lung disease (90%)
Hepatosplenomegaly (90%)
Hypothyroidism (50%)
Type 1 diabetes (25%)
Alopecia Universalis
(50%)
Lympthadenoptathy
(50%)
Hypothyroidism (40%)
Autoimmune Hepatitis (30%)
Enteropathy (20%)
Type 1 diabetes (10%)
Hypoparathyroidism
(80%)
Adrenal insufficiency
(88%)
Mucocutaneous
candidiasis (86%)
Gonadal insufficiency
(45%)
Type 1 Diabetes (13%)
Pernicious anaemia (13%)
Alopecia areata (45%)
Vitiligo (15%)
Autoimmune hepatitis
(15%)
Keratitis (28%)
Antibodies to type I
interferons (IFN-α or ω)
Autoimmune
thyroid(35%)
Autoimmune
haemolytic anaemia
(32%),
Thrombocytopenia
(15%),
Lympthadenopathy
(8%)
Eosinophilia
Very elevated IgE
FOXP3 expression in
CD4+ T Cells low
IgE typically normal
CD25 expression on T
cells absent or low (flow
cytometry)
Supplementary Figure 1
Western blot showing the expression of STAT3 protein in HEK-293 cells transfected with STAT3
contructs. HEK293 cells were lysed and protein extracts probed with anti-STAT3 antibody. β-actin was
used as a loading control. The experiment was repeated twice with similar results.
Supplementary Figure 2
Genotype-phenotype relationship in STAT3 mutations. The predicted effects of STAT3 mutations were
modelled in PDB structure 1bg1 (mouse STAT3/DNA complex) using SWISS-MODEL, and visualized in SwissPdbViewer. a) Overview of STAT3 dimer bound to DNA; STAT3 chains are shown in ribbon form, with residues
N646 (red) and N647 (green) shown as space-filling on the left chain only; DNA strands are shown as blue and
turquoise ribbons. b) as a), but expanded to show the proximity of residues N646 and N647 to both the DNAbinding and dimerization surfaces. c) Predicted molecular surfaces of wild-type STAT3 (wt), and mutants N646K,
N647D and N647I; surfaces are coloured 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 a) and b) to show relevant groups more clearly. The N646K mutation 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 mutation, previously reported as a loss-of function mutation 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 comparison, a different mutation at this position, N647I, has been
previously reported as an activating mutation 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 modelling in silico which predicts increased hydrophobicity
in this region (circled, N646I column, bottom row) compared to wild-type STAT3 or other variants.
References
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2
3
4
5
6
7
8
9
10
11
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