EBioMedicine 2 (2015) 744–749

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EBioMedicine
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Original Article

TYK2 Promoter Variant and Diabetes Mellitus in the Japanese
Seiho Nagafuchi a,b,h,⁎,1, Yumi Kamada-Hibio a,1, Kanako Hirakawa a, Nobutaka Tsutsu e, Masae Minami f,
Akira Okada g, Katsuya Kai a, Miho Teshima a, Arisa Moroishi a, Yoshikazu Murakami i, Yoshikazu Umeno j,
Yasushi Yokogawa k, Kazuhiko Kogawa k, Kenichi Izumi l, Keizo Anzai l, Ryuichi Iwakiri m,
Kazuyuki Hamaguchi n, Nobuhiro Sasaki e, Sakae Nohara e, Eiko Yoshida b, Mine Harada b, Koichi Akashi b,
Toshihiko Yanase o,p, Junko Ono o,p, Toshimitsu Okeda q, Ryoji Fujimoto q, Kenji Ihara d, Toshiro Hara d,
Yohei Kikuchi c, Masanori Iwase c, Takanari Kitazono c, Fumiko Kojima a, Suminori Kono r, Hironori Kurisaki a,
Shiori Kondo s, Hitoshi Katsuta a,b,haDepartment of Medical Science and Technology, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan
Department of Medicine and Biosystemic Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan
Department of Medicine and Clinical Sciences, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japand
Department of Pediatrics, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japane
Department of Diabetes and Metabolism, Fukuoka Red Cross Hospital, Fukuoka 815-8555, Japanf
Minami Masae Naika Clinic, Fukuoka 815-0071, Japang
Okada Naika Clinic, Fukuoka 812-0053, Japanh
Department of Internal Medicine, Sawara Hospital, Sawara, Fukuoka 819-0002, Japani
Yamaguchi Red Cross Hospital, Yamaguchi 753-8519, Japanj
Department of Diabetes, Oita Red Cross Hospital, Oita 870-0033, Japank
Department of Internal Medicine, Hamanomachi Hospital, Fukuoka 810-8539, Japanl
Departmtent of Hepatology, Diabetes and Endocrinology, Saga University, Saga 849-8501, Japanm
Department of Internal Medicine & Gastrointestinal Endoscopy, School of Medicine, Saga University, Saga 849-8501, Japann
Department of Medicine, School of Medicine, Oita University, 849-8501, Oita 879-5593, Japano
Department of Clinical Laboratory Medicine, School of Medicine, Fukuoka University, Fukuoka 814-0180, Japanp
Department of Diabetology and Metabolism, School of Medicine, Fukuoka University, Fukuoka 814-0180, Japanq
Department of Internal Medicine, Shinkokura Hospital, Kitakyushu 803-8505, Japanr
National Institute of Health and Nutrition, Tokyo 162-8636, Japans
Matsuyama Red Cross Hospital, Matsuyama 790-8524, Japanbca r t i c l e

i n f oArticle history:
Received 2 April 2015
Received in revised form 7 May 2015
Accepted 7 May 2015
Available online 9 May 2015
Keywords:
Tyrosine kinase 2 (TYK2)
Promoter variant
Diabetes mellitus
Virus
Polymorphisma b s t r a c t
Background: Recently, natural mutation of Tyrosine kinase 2 (Tyk2) gene has been shown to determine susceptibility to murine virus-induced diabetes. In addition, a previous human genome-wide study suggested the type 1
diabetes (T1D) susceptibility region to be 19p13, where the human TYK2 gene is located (19p13.2).
Methods: Polymorphisms of TYK2 gene at the promoter region and exons were studied among 331 healthy
controls, and 302 patients with T1D and 314 with type 2 diabetes (T2D) in the Japanese.
Findings: A TYK2 promoter haplotype with multiple genetic polymorphisms, which are in complete linkage
disequilibrium, named TYK2 promoter variant, presenting decreased promoter activity, is associated with anincreased risk of not only T1D (odds ratio (OR), 2.4; 95% conﬁdence interval (CI), 1.2 to 4.6; P = 0.01), but also
T2D (OR, 2.1; 95% CI, 1.1 to 4.1; P = 0.03). The risk is high in patients with T1D associated with ﬂu-like syndromeat diabetes onset and also those without anti-glutamic acid decarboxylase autoantibody.
Interpretation: The TYK2 promoter variant is associated with an overall risk for diabetes, serving a good candidate
as a virus-induced diabetes susceptibility gene in humans.
Funding: Ministry of Education, Culture, Sports, Science and Technology and of Health, Labor and Welfare of Japan.
© 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license(http://creativecommons.org/licenses/by/4.0/).

1. Introduction⁎ Corresponding author at: Department of Medical Science and Technology, Graduate
School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan.
E-mail address: nagafu_s@med.kyushu-u.ac.jp (S. Nagafuchi).
1
These authors contributed equally to this work.

Diabetes mellitus is on the rise worldwide, and is associated withimprovement in socioeconomic conditions, increasing wealth, higher
caloric and fat intake and lower physical activity (Scully, 2012;

http://dx.doi.org/10.1016/j.ebiom.2015.05.004
2352-3964/© 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

S. Nagafuchi et al. / EBioMedicine 2 (2015) 744–749

McCarthy, 2010). Accumulating evidence has also suggested the association of environmental factors such as toxins and viruses withdiabetes (Jonietz, 2012). However, the role of these environmental
factors in the development of diabetes is not yet fully understood.
Virus infection has long been considered to be a possible cause of type1 diabetes (T1D), as suggested by many clinical and experimental observations (Taylor, 2013; Coppieters et al., 2012). It was reported that several
viruses including coxsackie B virus, cytomegalovirus, varicella-zoster
virus, and rubella virus, were found in the pancreatic islets in patientswith severe fatal viral infections (Jenson et al., 1980), suggesting thatsystemic severe viral infections could lead to the pancreatic β-celldamage. Virus-induced diabetes is a more complex disease than previously thought, and is ascribed to diverse mechanisms that may leadto damage of the pancreatic β-cells (Coppieters et al., 2012). Thesemechanisms include direct virolysis, local inﬂammatory response, andtriggering of autoimmunity against β-cells (Coppieters et al., 2012).
However, the precise mechanisms of pancreatic β-cell damage
caused by viral infections remain to be determined, and host factorsthat control virus-induced diabetes have not been elucidated.
Accumulating evidence strongly suggests the contribution of enteroviruses, which belong to the picornavirus group, to the elevated risk of
diabetes (Tauriainen et al., 2011; Oikarinen et al., 2011; Tanaka et al.,2009). Since resistance to picornavirus infection has been shown to be
dependent on innate immunity (Ida-Hosonuma et al., 2005; Takeuchi
and Akira, 2009), the molecules regulating innate immune responses
are candidates for determining susceptibility to virus-induced diabetes(Kounoue et al., 2008; Nagafuchi et al., 2013). These include interferon
itself, interferon production and interferon receptor-mediated signaling pathway-associated molecules including pattern recognition
receptors (PRR) directed against pathogen-associated molecularpatterns (PAMPs) such as toll-like receptors (TLR) and intracellular
helicases such as retinoic acid-inducible gene I (RIG-I) and interferon
induced with the helicase C domain I (IFIH1) or melanocyte differentiation antigen (MDA)-5 (MDA-5/IFIH1) (Takeuchi and Akira, 2009).
Interferon-regulatory factors and interferon receptor-associated downstream molecules including JAK1, TYK2, STAT1 and STAT2 are also important with respect to serving as resistance against viral infections(Nagafuchi et al., 2013) (Supplementary Fig. 1). However, the exact
host factors that confer susceptibility to virus-induced diabetes
remain uncertain.
Since innate immunity plays a signiﬁcant role in the protection
against experimental encephalomyocarditis (EMC) virus (a picornavirus)-induced diabetes (Kounoue et al., 2008), it is suggested that intact
operation of the interferon signaling pathway may be important forresistance against virus-induced diabetes. It should be noted that thisexperimental virus-induced diabetes in mice is an excellent model as
rapid onset T1D including fulminant type, but not autoimmune diabetes(Imagawa et al., 2000; Nagafuchi et al., 2013). A separate study from ourgroup presented experimental evidence that the Tyk2 gene, an interferon receptor signaling pathway molecule, was responsible for encephalomyocarditis (EMC) virus-induced diabetes susceptibility in mice
(Izumi et al., 2015). Highly virus-induced diabetes-susceptible strains
such as SJL and SWR mice possessing a mutated Tyk2 gene, which is
associated with reduced expression of Tyk2 gene in pancreatic β-cells,
were prone to the development of diabetes caused by the diabetogenic
strain of EMC-D virus (Izumi et al., 2015). Interestingly, a humangenome-wide study suggested the T1D susceptibility region to be chromosome 19p13 (Mein et al., 1998), where the TYK2 gene was located(19p13.2) (Firmbach-Kraft et al., 1990). However, the exact responsible
gene has not yet been identiﬁed.
These observations suggest that the human TYK2 gene may be
associated with the risk for T1D and also confer a possible link withvirus-induced diabetes susceptibility in humans. We thus examined
the association of TYK2 gene polymorphisms with T1D and type 2
diabetes (T2D), focusing on association with ﬂu-like syndrome atdiabetes onset.

745

2. Methods
2.1. Subjects
We studied 947 Japanese patients and controls. Those include 302
patients with T1D, 314 patients with T2D and 331 healthy controls.
Among the 302 patients with T1D, 73 patients were associatedwith ﬂu-like syndrome at the onset. Clinical proﬁles of the Japanesepatients with T1D or T2D, and the healthy controls are presented inTable 1.
Patients were designated as T1D if fasting C-peptide was b 0.5 ng/ml
and they were in an insulin-dependent condition (IDDM), or as T2D if
fasting blood glucose levels were higher than 126 mg/dl and HbA1clevels exceeded 6.5% with non-insulin-dependent status (NIDDM).
Patients with T1D were also grouped according to their age at onset,
as 0 to 19 (0–19), 20 to 39 (20–39), 40 to 59 (50–59), and 60 to 79(60–79) years old. The study was conducted according to the guidelines
for human study and was approved by the ethical committee of the
Kyushu University, Graduate School of Medical Sciences (No. 433-00).
Written informed consent was obtained from all subjects including
T1D, T2D and healthy controls involved in this study.
2.2. Genotyping of TYK2 Gene
Genotyping had been performed to detect 25 exons and the putative promoter region, 1.3 kb upstream of start codon, of the TYK2
gene. TYK2 sequence reference was NCBI Reference Sequence: NG_
007872.1. PCR ampliﬁcation of the target genes, followed by the direct sequencing of the ampliﬁed gene, was conducted. A list ofprimers used to detect the polymorphisms of the TYK2 gene is presented in Supplementary Table 1.
2.3. TYK2 Promoter Variant Gene Analysis
To identify the TYK2 promoter variant, we used PCR analysis followed by direct sequencing to identify −930G N A and −929 T N A at thepromoter region, using the following primer sets: F:5′-GAA TCG CTTGAA TCC GGG AG-3′, and R:5′-ACC CTT CTT CTG TGC CAC AC-3′. Thus,
we present TYK2 promoter genotypes of wild type and variant type as
GT and AA, respectively.
2.4. Statistical Analysis
The genotype distribution between the cases and controls was statistically assessed by χ2 test. Odds ratio (OR) and 95% conﬁdence
Table 1
Characteristics of patients with T1D and T2D, and healthy controls.
Characteristics

Type 1 diabetesa Type 2 diabetes

Healthy controls

NumberAge (years) (range)

302
40.7 ± 17.3
(7–83)
37.4
8.9 ± 2.3
21.8 ± 3.1
27.8 ± 17.9
(0–73)
58.6

314
61.8 ± 11.8
(17–91)51.3
7.9 ± 1.6
23.2 ± 3.6
NAc

33143.4 ± 12.7
(18–71)53.5
5.2 ± 0.7
22.0 ± 3.3
NA

NTe

NT

Men (%)
HbA1c (%)b
BMI (kg/m2)
Age at diabetes onset(range)Anti-GAD Abd positive(%)

Values are means ± standard deviation.
T1D, type 1 diabetes; T2D, type 2 diabetes.
aPatients with T1D were diagnosed as they have fasting serum c-peptide level below
0.5 ng/ml.
b
HbA1c (%) was expressed as National Glycohemoglobin Standardization Program(NGSP) value.
c
NA: not available.
dAnti-GAD ab: anti-glutamic acid decarboxylase antibody (≥1.5 U/ml).
e
NT: not tested.

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S. Nagafuchi et al. / EBioMedicine 2 (2015) 744–749

interval (CI) were estimated by Woolf's method. Statistical analysis wasdone using Stata version 10 (Stata Corporation, College Station, Texas).
3. Results
3.1. Polymorphisms of Human TYK2 Gene
We ﬁrst screened human TYK2 gene polymorphisms in 22 patientswith T1D associated with ﬂu-like syndrome, suggestive of possible viral
infection, by PCR ampliﬁcation followed by direct sequencing. We
found seven polymorphisms: − 930G N A, − 929T N A and− 104A N C at the promoter region from transcription start point atexon 1; 1A N G, 62G N A and 63G N A at exon 1, which is an untranslated region; and 15597G/T at exon 8 with an amino acid substitution from valine to phenylalanine (V326F) (Fig. 1).
Among them, 1A N G (rs17000728), 62G N A (rs17000728) and
63G N A (rs2304258) at exon 1, and 15597G/T (V326F) at exon 8(rs2304256) have been already identiﬁed by the 1000 Genomes Project that included the Japanese population (The 1000 Genomes
Project Consortium, 2010, 2012). Because the polymorphisms at
the promoter region and exon 1 were in complete linkage disequilibrium in all 7 patients (Table 2), the haplotype was named TYK2 promoter variant.
3.2. Signiﬁcance of TYK2 Promoter Variant
We ﬁrst studied the association of the missense change at exon 8
with diabetes. We compared 244 patients with T1D, 255 patients with
T2D and 254 healthy controls, and found no measurable difference inthe genotype frequency between diabetic patients and healthy controls(Supplementary Table 2).

We further compared the prevalence of TYK2 promoter variant;
GT/AA and AA combined, and compared with wild type GT in 302
patients with T1D, 314 patients with T2D and 331 healthy controls(Table 1). Among them, 73 T1D patients had a ﬂu-like syndrome atdiabetes onset. The frequency of the TYK2 promoter variant was signiﬁcantly higher in patients with T1D (odds ratio (OR), 2.4; 95% conﬁdence
interval (CI), 1.2 to 4.6; P = 0.01), and also in patients with T2D (OR, 2.1;
95% CI, 1.1 to 4.1; P = 0.03), compared with healthy controls (Table 3).
Thus, the TYK2 promoter variant was more frequent in all patients withdiabetes compared with healthy controls (OR, 2.3; 95% CI, 1.2 to 4.1;
P = 0.009) (Table 3). The TYK2 promoter variant was associated with amore evident increase in risk of T1D patients associated with a ﬂu-like
syndrome (OR, 3.6; 95% CI, 1.5 to 8.5; P = 0.005) (Table 3). In addition,
the TYK2 promoter variant was signiﬁcantly more frequent among T1D
patients without anti-glutamic acid decarboxylase autoantibody (GAD)(OR, 3.3; 95% CI, 1.6 to 7.2; P = 0.002), but not among anti-GAD
autoantibody-positive patients (OR, 1.7; 95% CI, 0.8 to 3.9; P = 0.21)(Table 3).
It is thus suggested that the risk for diabetes conferred by the TYK2
promoter variant is distinct from autoimmunity against pancreatic βcells. We grouped TID associated with ﬂu-like syndrome and analyzed
the age at onset and anti-GAD antibody positivity, in association withTYK2 promoter variant. There was no statistical signiﬁcance in the age
at onset (P = 0.16), but has signiﬁcantly increased frequency in antiGAD antibody negative T1D (OR, 5.0; 95% CI, 1.9 to 13.2; P = 0.0005)(Supplementary Table 3), consistent with the observation of all T1D
patients. There was no gender difference in the frequency of TYK2 promoter variant, among patients with T1D (male, 13/113; 11.5%, female,
16/189; 8.5%; P = 0.39), and also T2D (male, 14/161; 8.7%, female,
13/153; 8.5%; P = 0.95). In the age-speciﬁc analysis on subjects with
T1D, we found that the TYK2 promoter variant haplotype was associatedwith a higher risk for diabetes in younger people aged 0 to 19 years (OR,Promoter Region

Exon 1

Exon 8-930 G>A -929 T>A-104 A>C

1 A>G

62 G>A 63 G>A

15597 G>T (V326F)

AAGTGG

GCAAA

ACAAG

AAGGGT

CAGTC

Wild

GT
AA

A
GG

GC

A
AA

AC

GG
AG

AA

G
GT

CA

TC

AA

C

G

AA

T

AAAAGG

GCCAA

ACGAG

AAAAGT

CATTC

HeteroHomoPositions of mutations were described from transcription start point at Exon1.
Fig. 1. Polymorphisms of TYK2 gene 22 patients with type 1 diabetes associated with ﬂu-like syndrome at the onset. Positions of mutations were described from transcription start point atexon 1.

S. Nagafuchi et al. / EBioMedicine 2 (2015) 744–749

747

Table 2
Screening of TYK2 gene polymorphism in 22 patients with T1D associated with ﬂu-like syndrome at diabetes onset.
Case no.

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22

Age at the onset

49
47
61
31
36
40
59
9
10
43
52
53
34
24
62
35
25
30
48
40
24
27

Sex

M
F
F
F
F
F
F
M
M
M
M
M
F
F
F
M
M
M
F
F
M
M

SNPs at promoter region

SNPs at exon 1

SNP at exon 8−930G N A

−929G N A

−104A N C

1A N G

62G N A

63G N A

HeteroHeteroHeteroHeteroHeteroHeteroHeteroHeteroHeteroHeteroHeteroHetero15597G N T

HeteroHeteroHetero
Homo

Homo

Homo

Homo

Homo

Homo

HeteroHeteroHeteroHeteroHeteroHeteroHeteroHeteroHeteroHeteroHeteroHeteroHeteroHeteroHeteroHeteroHeteroHeteroHeteroHeteroHeteroHeteroHeteroHeteroHeteroHeteroHeteroHeteroHeteroHeteroHomo

T1D, type 1 diabetes.
Hetero: heterozygous polymorphism.
Homo: homozygous polymorphism.

2.4; 95% CI, 1.1 to 5.4; P = 0.04) and 20 to 39 years (OR, 3.1; 95% CI, 1.4
to 6.9; P = 0.006), but not in older patients aged 40–59 years (OR, 1.6;
95% CI, 0.5 to 5.1; P = 0.62) or 60–79 years (OR, 1.2; 95% CI, 0.1 to 9.5;
P = 0.68) (Table 4).
Thus, the TYK2 promoter variant is associated with an increased riskof T1D with a younger age of onset.
In the age group of 20–39, we found no difference in the age at onset(P = 0.17), however, those with variant type are more associated with
anti-GAD negative people (OR, 5.1; 95% CI, 1.9 to 13.6; P = 0.0003), andalso with ﬂu-like syndrome at the onset (OR, 4.8; 95% CI, 1.4 to 15.49;
P = 0.022) (Supplementary Table 3), consistent with the observationin all T1D. Since obesity is an important risk for endocrinological disorders (Dwivedi et al., 2012), we also analyzed the association between
BMI and TYK2 promoter variant in T2D. There was no difference in
BMI between T2D with TYK2 promoter wild type gene and variant
type (P = 0.12) (Supplementary Table 4). In addition, there was increased TYK2 promoter variant rate at statistical signiﬁcance only in
non-obese T2D with less than 26 BMI (OR, 2.4; 95% CI, 1.2 to 4.8; P =0.01), but not obese T2D with more than 26 BMI (OR, 0.8; 95% CI, 0.2
to 3.7; P = 1.0) (Supplementary Table 4), suggesting that obesity is
not likely involved in the increased risk associated with TYK2 promoter
variant in T2D.
3.3. Promoter Activity of the TYK2 Promoter Variant
To determine the function of the TYK2 promoter variant gene, we
performed a luciferase assay, and found that the variant type promoter
showed signiﬁcantly reduced promoter activity (82.29 ± 0.03%;
P b 0.001) (Supplementary Fig. 2A). Similarly, there was a slight decrease in the interferon-induced expressions of TYK2 gene (mean ±SD of the relative expression; 0.59 ± 0.21) in peripheral blood mononuclear cells derived from diabetic patients with TYK2 promoter variant(n = 14), compared with those (0.71 ± 0.28) of patients with thewild type TYK2 gene (n = 17), while there was a mild increase in theinterferon-induced expressions of JAK1 gene (mean ± SD of the relative
expression; before stimulation; 0.74 ± 0.19 to 0.82 ± 0.17 after stimulation) in peripheral blood mononuclear cells derived from diabetic
patients with TYK2 promoter variant (n = 14), compared with those(before stimulation; 0.82 ± 0.21 to 0.84 ± 0.21 after stimulation) of

patients with the wild type TYK2 gene (n = 17) (Supplementary
Fig. 2B). The expression levels of ISGs, including PKR, OAS and MxA, induced by interferon stimulation in patients with TYK2 promoter variant
were also lower than those of patients with wild type ISGs (Supplementary Fig. 2C), which did not reach statistical signiﬁcance (all: P N 0.05).
These results suggest that the increased risk of developing diabetes conferred by the TYK2 promoter variant may be due to reduced TYK2 promoter activity accompanied by the decreased expression of the TYK2
gene and ISGs, while increased expression level of JAK1 gene on IFN
stimulation in patients with TYK2 promoter variant may play a complementary role for the deteriorated TYK2 gene expression to maintain ISGs
responses. Further investigation is required to clarify the inﬂuence ofTYK2 promoter variant on cytokine responses in humans.
4. Discussion
In the present study, based on our experimental evidence that thenatural susceptibility gene to EMC virus-induced diabetes was Tyk2 inmice (Izumi et al., 2015), we could extend those observations tohumans, with a TYK2 promoter variant which is associated with an
overall increased risk for diabetes in Japanese subjects, particularly inpatients with T1D associated with ﬂu-like syndrome at onset. In addition, a genome wide study had identiﬁed the T1D susceptibilityassociated region as 19p13 (Mein et al., 1998), where the TYK2 gene islocated (19p13.2)(Firmbach-Kraft et al., 1990). All these observations
taken together indicate that the TYK2 gene might be regarded as agood candidate for the virus-induced diabetes susceptibility gene inhumans. Surprisingly, the TYK2 gene promoter region variant wasmore frequent not only in subjects with T1D but also those with T2D.
If TYK2 promoter variant is actually associated with increased susceptibility to virus-induced diabetes, these results suggest that viral infection
may be one of the risk factors for developing T2D, which is consistent
with the concept that the accumulation of environmental insults willlead to clinical diabetes (Toniolo et al., 1980). Since TYK2 gene is also
associated with other several cytokine signals including IL-6, IL-10, IL12, and IL-23 (Strobl et al., 2011; Casanova et al., 2012; O'Shea et al.,2013), suggesting that deteriorated cytokine responses that can modulate immune/inﬂammatory reactions, alone or in combination, due to
TYK2 promoter variant, may also play a role to serve an increased risk

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S. Nagafuchi et al. / EBioMedicine 2 (2015) 744–749

Table 3
TYK2 promoter variant in patients with T1D and T2D, and healthy controls.
T1D

Genotype

Healthy control(n = 331)

No (%)

Flu-like syndrome
associated (n = 73)

All (n = 302)

No (%)

GT

317 (95.8)

273 (90.4)

GT/AA

14 (4.2)

28 (9.3)

ORc (95% CId)
1.00bNo (%)

0 (0.0)1 (0.3)eP-value

1.00

No (%)b

OR (95% CI)

No (%)b

164 (92.7)

3.6 (1.5−8.5)
1 (1.4)

0.01

Negative(<1.5 U/ml)(n = 125)

1.00

12 (6.8)

9 (12.3)
2.4 (1.2−4.6)

AA

Positive (≥1.5 U/ml)(n = 177)

OR (95% CI)

63 (86.3)

OR (95% CI)
1.00

109 (87.2)

No (%)b

16 (12.8)
1.7 (0.8−3.9)

1 (0.6)0.005

T2D (n = 314)

Anti-GAD antibody

a

1.00b

287 (91.4)
25 (8.0)

3.3 (1.6−7.2)
0 (0)

0.21

OR (95% CI)

2.1 (1.1−4.1)
2 (0.6)

0.002

0.03

T1D, type 1 diabetes; T2D, type 2 diabetes.
a
Symptoms of ﬂu-like syndrome include fever, chills, sore throat, muscle and joint aches, poor appetite, diarrhea, cough, and fatigue, suggestive of certain viral infections.
b
Referent.
cOR, odds ratio.
dCI, conﬁdence interval.
eHeterozygous (GT/AA) and homozygous (AA) variant genotypes combined (TYK2 promoter variant) versus homozygous wild genotype (GT) between the cases and healthy controls was
statistically assessed by χ2 test.

for diabetes. Accordingly, it has been indicated to have a close link
between inborn errors or polymorphisms of TYK2 gene and a wide spectrum of autoimmune diseases, inﬂammatory diseases, tumors, and obesity (Strobl et al., 2011; Casanova et al., 2012; O'Shea et al., 2013;
Derecka et al., 2012). Therefore unknown factors associated with the
TYK2 promoter variant other than viral infection may also contributeto increase the risk for diabetes. At least, as indicated in our study,
TYK2 promoter variant in T2D was associated with non-obese patients
but not with obesity.
Interestingly, TYK2 promoter variant was associated with a signiﬁcantly higher susceptibility to diabetes in anti-GAD antibody-negative
patients (OR, 3.3; P = 0.002), of which observation is consistent withTyk2 gene mutation dependent murine virus-induced diabetes that simulates non-autoimmune rapid onset and fulminant T1D without autoantibody production (Imagawa et al., 2000; Nagafuchi et al., 2013;
Izumi et al., 2015). It was reported that the Tyk2 gene played an important role not only in the interferon signaling pathway but also in the Th1
type immune response-associated IL-12-dependent signaling pathway(Shimoda et al., 2000). It was also reported that autoimmunity topancreatic β-cells, associated with the development of T1D, was mainly
dependent on Th1 type immune response (Haskins and Cooke, 2011).
These observations suggest that the human TYK2 gene promoter variant
may reduce the risk for the development of Th1 type-dependent
autoimmune reactivity to the pancreatic β-cells. In addition, if TYK2
promoter variant confer risk for the development of diabetes due toTable 4
TYK2 promoter variant genotypes in patients with T1D, with stratiﬁcation by the age of
onset.
Age at onset

0–19
20–39
40–59
60–79

T1D (n = 302)
Wild

HeteroHomo

104 (90.4%)
94 (87.9%)
56 (93.3%)
19 (95.0%)

10 (8.7%)
13 (12.1%)
4 (6.7%)1 (5.0%)1 (0.9%)0 (0%)
0 (0%)
0 (0%)ORa (95% CIb)

P-valuec

2.4 (1.1–5.4)
3.1 (1.4–6.9)
1.6 (0.5–5.1)
1.2 (0.1–9.5)

0.04
0.006
0.62
0.68

T1D, type 1 diabetes.
aOR, odds ratio.
b
CI, conﬁdence interval.
cHeterozygous and homozygous variant genotypes combined (TYK2 promoter variant)versus homozygous wild genotype in comparison with healthy controls (see Table 3).

increased susceptibility to viral-infection, possible induction of autoimmunity against pancreatic β-cells triggered by viral infections, which is awell-documented hypothesis (Fairweather and Rose, 2002; Stene et al.,2010), was not a major pathogenic mechanism, in TYK2 promoter
variant-associated susceptibility to type 1 diabetes. Since these data
have been obtained in the Japanese population, less prone to T1D thanother ethnic groups, the possible role of TYK2 promoter variant needs
to be veriﬁed in different populations.
In human cases the situation is highly different from that in experimental animals where mice have been infected with a virus and it ispossible to prove that infection is causing diabetes, however, accumulation of circumstantial evidence to identify the putative virus-induced
susceptibility gene in humans is important. It was reported that polymorphisms of the IFIH1 gene, which is an intracellular pathogen recognition receptor for picornavirus including enteroviruses, operating as an
inducer of interferon production (Takeuchi and Akira, 2009), is associated with risk or resistance for the T1D, serving possible virusinduced susceptibility gene in humans (Smyth et al., 2006; Nejentsev
et al., 2009). Since the outcome of virus-induced diabetes is inﬂuenced
by many factors including viral diabetogenicity and host susceptibility,
the discovery of other risk genes associated with virus-induced diabetes
in addition to IFIH1 and TYK2 genes is both possible and feasible. Unfortunately, at present, there is no appropriate assay system to prove thediabetogenicity of the virus infective for humans, fulﬁlling ‘Koch’s
postulate (Tabrah, 2011). Mouse models that are simulative of humanvirus-induced diabetes, with higher virus-induced diabetes susceptibility for use as an in vivo assay system to evaluate the diabetogenic potential of the possible viral agents that are infectious for humans. Mousestrains endowed with high susceptibility to picornavirus-induced diabetes may be used as in vivo models to evaluate the diabetogenicity ofcandidate human viruses.

Author Contributions
S.Na. designed the study, interpreted the data and wrote the manuscript. Y.K-H., K.Hi., K.Ka., M.T., A.M., E.Y., H.Ku, and H.Ka. performed thegenetic analyses. N.T., M.M., A.O., Y.M., Y.U., Y.Y., K.K., K.I., K.An., R.I.,
K.Ha., N.S., S.No., K.Ak., T.Y., J.O., T.O., R.F., K.I., T.H., M.H., Y.K., M.I., T.K.,
F.K., H.Ka., S.Na., and S.Kondo collected the human samples of the

S. Nagafuchi et al. / EBioMedicine 2 (2015) 744–749

patients and healthy controls, and analyzed the clinical data of thepatients. S.Kono performed the statistical analysis.
Conﬂict of Interest
There is no conﬂict of interest regarding this research.
Acknowledgments
The authors thank Itsuka Matsumoto, Ai Umei, Miki Kawano,
Makoto Matsuo, Kiyoka Nishibayashi, Keiko Kajishima, and Dr. Morio
Umeno at Sawara Hospital for their contribution in conducting thisresearch. We acknowledge Angela Koh, Khoo Teck Puat Hospital,
Singapore, and Dr. Chiri Nagatsuka for their help in the preparation of
the manuscript. The authors also thank Drs. Aida Kaoru, Tetsuro
Kobayashi, Nao Nishida and Takehiko Sasazuki for their helpful discussions. We also appreciate the technical support from the Research Support Center, Graduate School of Medical Sciences, Kyushu University.
This work was supported by grants (19209037 and 21659230) from
the Ministry of Education, Culture, Sports, Science and Technology anda grant (H22-Nanchi-097) from the Ministry of Health, Labor and Welfare of Japan. The funders had no role in study design, data collection,
data analysis, interpretation, or writing of the report.
Appendix A. Supplementary Data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.ebiom.2015.05.004.
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Supplementary Information
Supplementary Methods Summary
Promoter Analysis of the TYK2 Gene
The PCR-amplified TYK2 promoter fragment, with either wild or variant typesequences, was cloned into a pGL4.17[luc2/Neo] vector (Promega). Insert was preparedfrom wild type TYK2 or homozygous TYK2 promoter variant by PCR amplification
using forward primer 5’-AAAGCTAGCAGCTGCCCTGTGAGGAGGC-3’ and reverse
primer 5’-AAAAAGCTTTCCCCGCGGCTTCTTCCTGA-3’, which led to a 1572bp
product. Luciferase assay was conducted by transfection of vectors to 293T cells with
24-well plates. Luciferase activity was measured 24 hours after transfection in 293T
cells using a dual luciferase assay kit (Promega Corporation, Madison, WI). The
experiments were repeated five times.
Expression of TYK2 gene and interferon stimulated genes (ISGs), including PKR, OSR
and MxA gene	  Patients with type 2 diabetes, possessing either TYK2 wild type or promoter variant,were studied for the expression of TYK2 gene, JAK1 gene and interferon-stimulated
genes before and after IFN-β stimulation. 14 patients with type 2 diabetes (age,65.1±10.8; HbA1c, 7.3±0.8%) carrying heterozygous (n=12) and homozygous (n=2)
TYK2 promoter variant, and 17 patients with type 2 diabetes (age, 71.8±9.9; HbA1c,
7.1±0.6%) carrying wild type TYK2 promoter were studied. The data are expressed asmeans±standard deviations.
PBMCs were isolated by LSM (MP BIOMEDICALS, Ohio, USA) from patients.
PBMCs were stimulated with IFN-β (500U/ml) (SIGMA-ALDRICH, Missouri, USA)for 12h, after which total RNA was extracted using ISOGEN (Wako Chem., Tokyo).
cDNA was synthesized from the RNA template (1 µg) with High-Capacity cDNA
Reverse Transcription Kits (Applied Biosystems) according to the manufacturer
protocol. Quantitative PCR was carried out by using an ABI 7500 real-time PCR systemwith Power SYBER green Master Mix (Applied Biosystems). The PCR was set up
under the following thermal cycling conditions: 50℃ 2min, 95℃ 10min, followed by
40 cycles of 95℃ 15sec, and 63℃ 1 min. Fluorescence signals were collected by themachine using the extension phase of each PCR cycle. The threshold cycle value wasnormalized to that of β-actin. The qPCR was performed by using the following
primer pairs: for human TYK2 gene, 5’-TGGCATGAATCCTCGGGAAC-3’
and5’-CATGCTTGCCCTGCTCAAAG-3’;
JAK1
gene,
5’-CTACAGTCTGCACGGTTCGGA-3’ and5’-CGATCGAAACTCAGTTGGCTC-3’;
Proteinkinase
R
(PKR)
gene,
5’-TCTGACTACCTGTCCTCTGGTTCT-3’
and5’-GCGAGTGTGCTGGTCACTAAAG-3’; 2'-5' oligoadenylate synthetase (OAS) gene,
5’-ACCTGGTTGTCTTCCTCAGTCC-3’
and5’-GAGCCTGGACCTCAAACTTCAC-3’; myxovirus resistance A (MxA) gene,
5’-TTCGGCTGTTTACCAGACTCC-3’ and 5’-CAAAGCCTGGCAGCTCTCTAC-3’;β-actingene,
5’-GCACCACACCTTCTACAATGAGC-3’
and5’-GGATAGCACAGCCTGGATAGCAAC-3’.
1

The experiments were repeated three times. The relative mRNA level was expressed as
fold change relative to the value of the corresponding healthy non-diabetic control.
Statistical analysis was done by Student’s t-test.

2

Supplementary Figures

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Supplementary Figure 1. Type 1 interferon (IFN-α/β) production in response to
putative diabetogenic virus infection and IFN signaling pathway.
JAK1 and TYK2 are reciprocal IFN receptor-associated molecules, mediating thedownstream signal to induce IFN-stimulated genes (ISGs) to resist against viral
infection. (modified from Diabetes and Viruses 2013, Springer Science-Media, p41,
Fig.5.3)

3

Supplementary Fig. 2. 

TYK2!
Before"IFNMβ""S,mula,on

Rela,ve"expression"level"of!TYK2

2"

100"
80"
60"
40"
20"



1.5"

1"

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0"

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Wild
Variant Control""wild%
mutant%control%
Nega,ve

PKR!

1.5"

Before"Before"IFNMβ""S,mula,on

†

0.5"

Wild"""""Variant"""""Wild"""""Variant"

Before"

AQer

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2"

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AQer"IFNMβ""S,mula,on

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1.5"

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0"

Wild"""""Variant"""""Wild"""""Variant"

OAS!

AQer

1"

0"

2"

Wild"""""Variant"""""Wild"""""Variant"Rela,ve"expression"level"of!OASRela,ve"expression"level"of!PKR

C."
2"

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AQer"IFNMβ""S,mula,on

Rela,ve"expression"level"of!MxARela,ve"Ac,vity"[%]

B."

*

120"

rela,ve"expression"level"of"JAK1

Promoter"Ac,vity"

A."

Before"
4"

MxA!

AQer

†††

3"

2"

1"

0"

Wild"""""Variant"""""Wild"""""Variant"

Supplementary Figure 2. Promoter assay of TYK2 promoter variant andexpression level of TYK2, JAK1, and interferon-stimulated genes.
Diabetic patients with heterozygous TYK2 promoter variant type were compared withthose with wild type TYK2 gene. A. Promoter activity of TYK2 promoter variant wasassessed by the luciferase assay. Relative activity of the luciferase assay of TYK2
promoter activity was expressed as percent, compared with that of wild type (100%).
Mutated TYK2 promoter variant showed significantly reduced promoter activity(82.6%±0.21) (n=9). (*P<0.001) B. Relative expression level of TYK2 gene induced byinterferon-β (IFN-β) stimulation in patients with diabetes possessing heterozygous
TYK2 promoter variant (n=12) (●: before stimulation, ▲: after IFN-β stimulation) andhomozygous patients (n=2) (●: before stimulation, ▲: after IFN-β stimulation) (before
stimulation, 0.58±0.21; after stimulation, 0.59±0.21) compared with those with wild
type gene (n=17) (○: before stimulation; 0.61±0.18, △: after IFN-β stimulation;
0.71±0.28). (#P=0.17). Relative expression level of JAK1 gene induced by interferon-β(IFN-β) stimulation in patients with diabetes possessing heterozygous TYK2 promoter
variant (n=12) (●: before stimulation, ▲: after IFN-β stimulation) and homozygous
patients (n=2) (●: before stimulation, ▲: after IFN-β stimulation) (before stimulation,
0.74±0.19; after stimulation, 0.82±0.17) compared with those with wild type gene(n=17) (○: before stimulation; 0.82±0.21, △: after IFN-β stimulation; 0.84±0.21)(##P=0.781). C. Relative expression level of ISGs induced by interferon-β (IFN-β)
stimulation in patients with diabetes possessing heterozygous TYK2 promoter variant
4

(n=12) (●: before stimulation, ▲: after stimulation) and homozygous patients (n=2)(●: before stimulation, ▲: after stimulation) compared with those with wild type gene(n=17) (○: before stimulation, △: after stimulation). Results of relative expressions of
ISGs are shown in patients with heterozygous and homozygous TYK2 promoter variants.
Before stimulation: PKR, 0.57±0.15; OAS, 0.58±0.20; MxA, 0.40±0.17. After
stimulation: PKR, 0.79±0.27; OAS, 0.96±0.50; MxA, 0.77±0.54. In patients with wild
type TYK2 gene: before stimulation: PKR, 0.64±0.19; OAS, 0.62±0.33; MxA, 0.64±0.51.
After stimulation: PKR, 0.79±0.18; OAS, 0.99±0.59; MxA, 1.02±0.75.	
  The data areexpressed as means ± standard deviations. (†P=0.94, ††P=0.87, †††P=0.30)

5Supplementary Table 1. Set of primers for amplification of TYK2 gene.
Forward

ReversePromoter Region

5’-GCCAGACCCCATCTCTACAAA-3’

5’-GGGAACACAAGCTCGAACC-3’

Exon 15’-AATCGCGGCTGAGTGACGAATG-3’5’-GACCCAGACCCAGCTTTGAAGA-3’

Exon 25’-CTGGACATAAACTCTCCTAGGC-3’5’-GACCATCTTGACCAACATGGTG-3’

Exon 35’-GTGGGTGGAAGGTTGAAGAG-3’!5’-GTGGATAGACGGATGGATGG-3’

Exon 45’-GGCTGACGGTAGCAAATGAC-3’

!5’-CTGGGGCTTAGCACAGAGTC-3’

Exon 55’-GAAGCTGGTCTGACTCTGTGC-3’

5’-GCCCCTAAGTCTCCCACAA-3’

Exon 65’-CTCTGGGCTAGAGAGGAACG-3’

5’-GTCTACCCTGGCTCCCAGAT-3’

Exon 75’-ACCTGGCTAGTGTGCCTGTT-3’

5’-TCAGAGGCTAGGGTCAAGGA-3’

Exon 85’-GGAGGTATAAACGGGCATTG-3’

5’-GGAAATAGCCGTCCACCAG-3’

Exon 95’-GTAGGGGCTGGGCTAGGG-3’5’-CCCCTAGGGCTCACAGTCTA-3’

Exon 105’-GGGTATGGGTCCAGAGTGG-3’

5’-GCAGAGGTGGGAGCAGTAAG-3’

Exon 115’-TACCGCCTGATCCTCACAGT-3’

5’-GCAGGCATCAAGTCATGGAG-3’

Exon 125’-GTGGGATGTGGCATCTCTCC-3’

5’-TGAAAGTTAGCAGCTGATCTCC-3’

Exon 135’-TGGGAGATCAGCTGCTAACTT-3’5’-GCCACCTCCTCCACAGAC-3’

Exon 145’-GTGTGTCCGTGGAGGAGGT-3’5’-GAGGGTTGGGGTACAGATCA-3’

Exon 15

5’-ATCCAGAGGGCAGAAGCAG-3’5’-AGGCTGGTCTCGAACTCCTG-3’

Exon 165’-GTTGGCGTCTGTGCCTCT-3’5’-GCGAAAGGAGCAGGGGAAG-3’

Exon 175’-CTTCCCCTGCTCCTTTCAC-3’

5’-AGAAGGGATGCAGCTTTGAG-3’

Exon 18

5’-GACTCCTCTGGGTCCCTTTC-3’

5’-CCTCTCGTGCGCTATAGGCA-3’

Exon 195’-TTTGTGACTCCCAAGTGTGG-3’5’-CTCAACCCCCCAAACTCCTTC-3’

Exon 20

5’-CACCCACGCTCTAACCACGC-3’

5’-TGGTGCAGGGATTGGGGAGG-3’

Exon 215’-CTCTGCTGGGCTCAAGGTAG-3’

5’-CCCAAGCTGAAGAGGAAGG-3’

Exon 225’-CTCCTGGCTGCTCAGGTC-3’

5’-CTGGGATCATGCCCTATCAT-3’

Exon 235’-GATCCCCAAGCCCTCAGT-3’5’-CCCAGCCTATGCCTTTCTAA-3’

Exon 24

5’-GCTGGGATTACAGGCATGAG-3’

5’-CCCTCTCCACAGCAGGATAG-3’

Exon 255’-CCTTTGTCTTTCCCTGACCC-3’

5’-CAGGGCTGCCATTGTGCCTC-3’

6

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