SUPPLEMENTARY INFORMATION EARLYONSET MULTIORGAN AUTOIMMUNITY CAUSED BY ACTIVATING GERMLINE

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Supplementary Information

Supplementary Information

Early-onset Multi-Organ Autoimmunity Caused by Activating Germline Mutations in STAT3

Sarah E. Flanagan,¹^,¹³Emma 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,⁴John Barton,⁵ Kaarina Heiskanen,⁶ Tarja Heiskanen-Kosma,⁷Merja Kajosaari,⁸Nuala P. Murphy,⁹ Tatjana Milenkovic,¹⁰ Mikko Seppänen,¹¹ Åke Lernmark,⁴ Timo Otonkoski,⁶Satu Mustjoki,³ Juha Kere,^2,12 Noel G. Morgan,¹Sian Ellard¹ and Andrew T. Hattersley¹

¹ Institute of Biomedical and Clinical Science, University of Exeter Medical School, Exeter, EX2 5DW, UK

²Folkhälsan Institute of Genetics, and Research Programs Unit, Molecular Neurology, University of Helsinki, Helsinki, Finland

³Hematology Research Unit Helsinki, Department of Hematology, University of Helsinki and Helsinki University Central Hospital Cancer Center, Helsinki, Finland

⁴Department of Clinical Sciences, Lund University/CRC, Skåne University Hospital SUS, Malmö, Sweden

⁵Bristol Royal Hospital for Children, Upper Maudlin Street, Bristol, BS2 8BJ, UK

⁶Children’s Hospital, Helsinki University Central Hospital and Research Programs Unit, Molecular Neurology, University of Helsinki, Helsinki, Finland

⁷Department of Pediatrics, Kuopio University Hospital, Helsinki, Finland

⁸Children’s Hospital, Helsinki University Central Hospital, Helsinki, Finland

⁹Department of Diabetes and Endocrinology, Children's University Hospital, Temple St., Dublin 1, Ireland

¹⁰Department of Endocrinology, Institute for Mother and Child Health Care of Serbia ‘Dr Vukan Cupic’, Belgrade, Serbia

¹¹Immunodeficiency Unit, Division of Infectious Diseases, Helsinki University Central Hospital, Helsinki, Finland

¹²Department of Biosciences and Nutrition, and Center for Innovative Medicine, Karolinska Institutet, Hälsovägen 7, 14183 Huddinge, Sweden

¹³These authors contributed equally to this work

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 site-directed 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 10⁵ 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	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

Supplementary Table 2: Features of 25 individuals with multiple early onset-autoimmune disease sequenced for STAT3 mutations

Patient	Early-onset diabetes	Autoimmune Enteropathy	Autoimmune pulmonary disease	Autoimmune thyroid dysfunction	Autoimmune joint disease	Dental anomalies	STAT3 mutation identified
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

Supplementary Table 3: STAT3 primers for Sanger sequencing

Exon	Forward Primer Sequences (M13 tailed) All primers start 5’ TGTAAAACGACGGCCAGT	Reverse Primer Sequences (M13 tailed) All primers start 5’ CAGGAAACAGCTATGACC
2	CCCCAGAGCATCTTTATCCC	CTCATTTTCCCCATCACCTG
3	GGGTTATAGCATCAGGTTTGC	AAGTATACAGAGCTTTGAGAAAGGG
4	TAGTAACGACCTCCCCTTCG	TCTGTTGGATTCTTTTGGTGG
5	TTCCCTTCCTCTTGTGATGG	CAAGAGAAGGCTCCCTGTTG
6	AACAGGGAGCCTTCTCTTGG	ATGACCAGGCTCCTTTGAGG
7	GGAGGTACGGGTCCTCAAAG	CAACTCCAGAGCAGGAACTTCT
8	ATTTCAGCGTCTTGTGGCAG	GCTAAATTTGAATATGGAAAAGTCC
9	TTTTCAGCATCCACCCAAC	GGAAAGAGAAGATGGGCTCAC
10	GGTAATTTAGCATCCTTGTCCC	ATGGCAACAAATTTCAACCC
11	GACAGCTTGGCCTATTTACCTG	TGTCCACAAAATGAAGATCTCTG
12	TGCGCTGATCAACTGTAACTG	ATTCCCACATCTCTGCTCCC
13	ATTCCCACATCTCTGCTCCC	TGCGCTGATCAACTGTAACTG
14	ATGGAAGAATCCAACAACGG	GTTCATGTCACTTTGGCCTG
15	TGCTGCTTAGACTGGTCTCG	CCCCTGTACGTAGCCTCTCA
16	CACTCCTCGCCTAGAGTTGG	GTCCTCGCTTGGTGGTG
17	AACATGCTGACCAACAATCC	GCCTTGCTCAGGAAAGAAAC
18F	AAATCCTCAGGCCCGTCTAC	CCTTCAAAGATGTGAAAGCTG
18R	CCTTCAAAGATGTGAAAGCTG	AAATCCTCAGGCCCGTCTAC
19	CTGAACTCTTGGTCCAGCG	AAAGCCCATGATGTACCTGG
20	GCTGGCAAGGGCTTCTC	AAGCAAACCAATCCTTCAGC
21	CACTACAATTCTTTCCCATAAGGAG	AACAGGGTGTTCAGGGTCTC
22	TAAATGAGGGCAGACAACCC	TCAAACTCTGGTCTCCAACAG
23	AGCCCCTGGGCTATGTTTAG	TCTCTTTTGGAAAGCAAAGCTC
24	TCCAGGGAGGAGGGTAAATC	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

Table of clinical features	PATIENT 1	PATIENT 2*	PATIENT 3	PATIENT 4	PATIENT 5
Mutation	p.Thr716Met (c.2147C>T)	p.Lys392Arg (c.1175A>G)	p.Asn646Lys (c.1938C>G)	p.Asn646Lys (c.1938C>G)	p.Lys658Asn (c.1974G>C)
Sex	Female	Female	Male	Male	Female
Current Age (yrs)	6	15	6	3	17
Birth weight (SDS)	-1.59	-5.81	-2.70	-1.59	-1
Growth (Height SDS)	-2.33	-6.63 Delayed puberty	-1.47	-2.05	-4.00 Delayed puberty
DIABETES insulin doses HbA1c
Age at Diagnosis (Wks)	2	0	3	43	Not diabetic
GAD65 autoantibodies	35 RU (<34)	40.3 RU (<5.36)	5 (<34)	31.9 U/mL (<1.0)	Negative
I-A2 autoantibodies	0 (<5)	0.08 (<0.43)	0(<5)	9.5 U/mL (<5)	Negative
ZnTn8 autoantibodies	7 (<60)	Not tested	11(<60)	Not tested	Not tested
IAA autoantibodies	Not tested insulin treated	3224 RU(<1.56)	Not tested insulin treated	Not tested insulin treated	Negative
ICA	Negative	28 JDFU(<2.5)	Negative	Not tested	Negative
Pancreatic Exocrine Insufficiency	Fecal elastase 166mg/ g faeces (>200)	On enzyme replacement therapy- Low Fecal elastase	NO	NO	NO
T1D HLA Type†	High Risk DRB103 -DQB102/ DRB103 -DQB102	High Risk DRB104 -DQB10302	Low Risk DQA101-DQB105/ DQA101-DQB10602	Low Risk DQA103-B103:02/A101-B106:02	Low risk
Current Insulin dose (U/Kg/day)	1.8	1.5	0.76	0.8	N/A
Current HbA1c (mmols/mol)	61	70	68	69	Not tested
ENTEROPATHY
Type	Coeliac disease	Coeliac-disease	NO	NO	Autoimmune enteropathy
Onset of symptoms (months)	17 months	<12 months	N/A	N/A	<12 months
Coeliac HLA type	High Risk DR52-DR3-DQ2	High risk DQ8	Not tested	Not tested	Low risk
Anti TtG autoantibodies	TtG IgA 20.47 U/mL (<10) TtG IgG > 200 U/mL (<10)	Not tested	<1.0 (<10)	2.8 (<7)	Not tested
Endomysial/gliadin autoantibodies	Not tested	Positive Endomysial & gliadin autoantibodies	Not tested	Not tested	Positive for gliadin autoantibodies
Responsive to gluten free diet	YES	YES	N/A	N/A	NO
OTHER AUTOIMMUNE FEATURES
Eczema	NO	Mild atopic	YES	YES	Non-specific dematitis
Pulmonary manifestations	None	Desquamative interstitial pneumonitis	None	None	Asthma Recurrent URTI
Other features	Primary hypothyroidism (TPO antibodies 224 U/mL (<20). Lipoatrophy at sites of insulin injection	None	None	Juvenile arthritis, severe dry eyes	Progressive macular edema & vision loss. Kawasaki disease-like episodes
HEMATOLOGICAL FEATURES
	NO	T-cell LGL leukemia. Autoimmune cytopenias.	Hb 10.0g/dl nd MCV 64.7fl (73-89) MCH 20.4pg (24-30)	NO	Lymphadenopathy & splenomegaly. Autoimmune cytopenias
DENTAL ANOMALIES
	NO	Dentures	NO	NO	Delayed eruption of primary teeth
IMMUNODEFICIENCY
Ig E	<2.0 (<100kU/L)	-	<2.0 (<100kU/L)	<2.0 (<100kU/L)	0.7 (3.9-10)
Eaosinophils	1.3% (<6%)	-	0.30 x10⁹/L (<0.7x10⁹/L)	0.28 x10⁹/L (0.08-1.1x10⁹/L)	-
Infection susceptibility	NO	Recurrent bacterial LRTI, IVIG from 12 y/o	NO	NO	Recurrent bacterial URTI, 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 in³. †HLA typing was undertaken on patients 1-4 using previously described methods⁴.

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	STAT3 polyautoimmunity	APS1	IPEX	CD25 deficiency	ITCH deficiency
Gene	STAT3	AIRE	FOXP3	IL2RA	ITCH
Inheritance	Dominant	Recessive	Recessive	Recessive	Recessive
Number of patients reported	5 patients (5 families)	>150	>70	4 patients (4 families)	10 patients (1 family)
References	This paper	Peterson, & Peltonen (2005)⁷	d'Hennezel, et al (2012)⁸ Wildin et al(2002)⁹	Bezrodnik, et al (2013)¹⁰	Lohr et al. (2010)¹¹
Common clinical features (present in >50% of patients	Type 1 diabetes (80%) Enteropathy (60%) Short stature (100%)	Hypoparathyroidism (80%) Adrenal insufficiency (88%) Mucocutaneous candidiasis (86%)	Enteropathy (100%) Eczematous Dermatitis (70%) Type 1 diabetes (67%)	Enteropathy (100%) Recurrent persistent viral infection(100%) Eczema (75%)	Developmental delay (100%) Macrocephaly(90%) Dysmorphic facies (90%) Chronic lung disease (90%) Hepatosplenomegaly (90%)
Additional clinical features	Autoimmunoe thyroid (40%) Juvenile Chromic Arthritis (20%) Fibrotic lung disease (20%)	Gonadal insufficiency (45%) Type 1 Diabetes (13%) Pernicious anaemia (13%) Alopecia areata (45%) Vitiligo (15%) Autoimmune hepatitis (15%) Keratitis (28%)	Autoimmune thyroid(35%) Autoimmune haemolytic anaemia (32%), Thrombocytopenia (15%), Lympthadenopathy (8%)	Hypothyroidism (50%) Type 1 diabetes (25%) Alopecia Universalis (50%) Lympthadenoptathy (50%)	Hypothyroidism (40%) Autoimmune Hepatitis (30%) Enteropathy (20%) Type 1 diabetes (10%)
Other laboratory or diagnostic features	Normal eosinophil count Low-normal IgE	Antibodies to type I interferons (IFN-α or ω)	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 INFORMATION EARLYONSET MULTIORGAN AUTOIMMUNITY CAUSED BY ACTIVATING GERMLINE

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 Swiss-PdbViewer. 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 DNA-binding 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.

SUPPLEMENTARY INFORMATION EARLYONSET MULTIORGAN AUTOIMMUNITY CAUSED BY ACTIVATING GERMLINE

References

1 Lango Allen, H. et al. Nature genetics 44, 20-22, (2012).

2 Ellard, S. et al. Diabetologia 56, 1958-1963,(2013).

3 Otonkoski, T. et al. Diabetologia 43, 1235-1238,(2000).

4 Sjoroos, M. et al BioTechniques 18, 870-877 (1995).

5 Russell MA et al. Islets 5, 95-105 (2013)

6 Gambineri, E. & Torgerson, T. R. et al CMLS 69, 49-58, (2012).

7 Peterson, P. & Peltonen, L.. Journal of autoimmunity 25 Suppl, 49-55,(2005).

8 d'Hennezel, E. et al Journal of medical genetics 49, 291-302, (2012).

9 Wildin, R. S. et al Journal of medical genetics 39, 537-545 (2002).

10 Bezrodnik, L. et al Clinical and experimental immunology (2013).

11 Lohr, N. J. et al. American journal of human genetics 86, 447-453 (2010).

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