Boisson-Dupuis et al., Sci. Immunol. 3, eaau8714 (2018) 21 December 2018
SCIENCE IMMUNOLOGY
|
RESEARCH ARTICLE
1 of 19
TUBERCULOSIS
Tuberculosis and impaired IL-23–dependent IFN-
immunity in humans homozygous for a common TYK2
missense variant
Stéphanie Boisson-Dupuis
1,2,3
*
†
, Noe Ramirez-Alejo
1†
, Zhi Li
4,5‡
, Etienne Patin
6,7,8‡
, Geetha Rao
9‡
,
Gaspard Kerner
2,3‡
, Che Kang Lim
10,11‡
, Dimitry N. Krementsov
12‡
, Nicholas Hernandez
1
, Cindy S. Ma
9,13
,
Qian Zhang
1,14
, Janet Markle
1
, Ruben Martinez-Barricarte
1
, Kathryn Payne
9
, Robert Fisch
1
,
Caroline Deswarte
2,3
, Joshua Halpern
1
, Matthieu Bouaziz
2,3
, Jeanette Mulwa
1
, Durga Sivanesan
15,16
,
Tomi Lazarov
17
, Rodrigo Naves
18
, Patricia Garcia
19
, Yuval Itan
1,20,21
, Bertrand Boisson
1,2,3
, Alix Checchi
2,3
,
Fabienne Jabot-Hanin
2,3
, Aurélie Cobat
2,3
, Andrea Guennoun
14
, Carolyn C. Jackson
1,22
,
Sevgi Pekcan
23
, Zafer Caliskaner
24
, Jaime Inostroza
25
, Beatriz Tavares Costa-Carvalho
26
,
Jose Antonio Tavares de Albuquerque
27
, Humberto Garcia-Ortiz
28
, Lorena Orozco
28
,
Tayfun Ozcelik
29
, Ahmed Abid
30
, Ismail Abderahmani Rhorfi
30,31
, Hicham Souhi
30
,
Hicham Naji Amrani
30
, Adil Zegmout
30
, Frédéric Geissmann
17
, Stephen W. Michnick
15
,
Ingrid Muller-Fleckenstein
31
, Bernhard Fleckenstein
31
, Anne Puel
1,2,3
, Michael J. Ciancanelli
1
,
Nico Marr
14
, Hassan Abolhassani
10,32
, María Elvira Balcells
33
, Antonio Condino-Neto
27
,
Alexis Strickler
34
, Katia Abarca
35
, Cory Teuscher
36
, Hans D. Ochs
37
, Ismail Reisli
38
, Esra H. Sayar
38
,
Jamila El-Baghdadi
39
, Jacinta Bustamante
1,2,3,40§
, Lennart Hammarström
10,11,41§
, Stuart G. Tangye
9,13§
,
Sandra Pellegrini
4,5§
, Lluis Quintana-Murci
6,7,8§
, Laurent Abel
1,2,3||
, Jean-Laurent Casanova
1,2,3,42,43
*
||
Inherited IL-12R1 and TYK2 deficiencies impair both IL-12– and IL-23–dependent IFN- immunity and are rare
monogenic causes of tuberculosis, each found in less than 1/600,000 individuals. We show that homozygosity for
the common TYK2 P1104A allele, which is found in about 1/600 Europeans and between 1/1000 and 1/10,000 indi-
viduals in regions other than East Asia, is more frequent in a cohort of patients with tuberculosis from endemic
areas than in ethnicity-adjusted controls (P = 8.37 × 10
−8
; odds ratio, 89.31; 95% CI, 14.7 to 1725). Moreover, the
frequency of P1104A in Europeans has decreased, from about 9% to 4.2%, over the past 4000 years, consistent
with purging of this variant by endemic tuberculosis. Surprisingly, we also show that TYK2 P1104A impairs cellular
responses to IL-23, but not to IFN-, IL-10, or even IL-12, which, like IL-23, induces IFN- via activation of TYK2 and
JAK2. Moreover, TYK2 P1104A is properly docked on cytokine receptors and can be phosphorylated by the proximal
JAK, but lacks catalytic activity. Last, we show that the catalytic activity of TYK2 is essential for IL-23, but not IL-12,
responses in cells expressing wild-type JAK2. In contrast, the catalytic activity of JAK2 is redundant for both IL-12
and IL-23 responses, because the catalytically inactive P1057A JAK2, which is also docked and phosphorylated,
rescues signaling in cells expressing wild-type TYK2. In conclusion, homozygosity for the catalytically inactive
P1104A missense variant of TYK2 selectively disrupts the induction of IFN- by IL-23 and is a common monogenic
etiology of tuberculosis.
INTRODUCTION
About a quarter of the world’s population is infected with Mycobacterium
tuberculosis, but this bacterium causes tuberculosis in less than 10% of
infected individuals, generally within 2 years of infection (a situa-
tion referred to here as primary tuberculosis) (1–3). In the countries
in which tuberculosis is highly endemic, primary tuberculosis is
particularly common in children, who often develop life-threatening
disease (4–6). Clinical and epidemiological studies have long suggested
that tuberculosis in humans has a strong genetic basis (7–9). Auto-
somal recessive (AR) complete interleukin-12 receptor 1 (IL-12R1)
and tyrosine kinase 2 (TYK2) deficiencies are the only two inborn
errors of immunity reported to date to underlie primary tuberculosis
in otherwise healthy patients in two or more kindreds (10–17). Cells
from patients with IL-12R1 deficiency do not respond to IL-12 or
IL-23 (12, 18–24). These patients are susceptible to weakly virulent
mycobacteria, such as the Bacille Calmette-Guérin (BCG) vaccine and
environmental species [Mendelian susceptibility to mycobacterial
disease (MSMD)], to the more virulent species M. tuberculosis, and
more rarely to Candida albicans (20, 25). They are prone to MSMD
and tuberculosis because they produce too little interferon- (IFN-)
(7, 12, 26, 27) and, in some cases, to chronic mucocutaneous candi-
diasis (CMC) because they produce too little IL-17A/F (28–32).
In patients with TYK2 deficiency, cellular responses to IL-12 and
IL-23 are severely impaired, but not abolished (10, 33–35). These
patients are, thus, also prone to MSMD and tuberculosis, although
probably with a lower penetrance than for IL-12R1 deficiency, be-
cause they display residual responses to IL-12 and IL-23. They do
not seem to be susceptible to C. albicans, which may merely reflect
the lower penetrance of candidiasis and smaller number of patients,
when compared with IL-12R1 deficiency. However, unlike patients
with IL-12R1 deficiency, they are susceptible to viral diseases due
to the impairment of their responses to IFN-
/ (10, 36). In vitro,
their cells respond poorly to IL-10, but this defect, which is not
observed in patients with IL-12R1 deficiency, is clinically silent
(10, 37, 38). Both IL-12R1 and TYK2 deficiencies are caused by
rare or private alleles, accounting for each deficiency being found in
Copyright © 2018
The
Authors, some
rights reserved;
exclusive licensee
American Association
for the Advancement
of Science. No claim
to original U.S.
Government Works
Boisson-Dupuis et al., Sci. Immunol. 3, eaau8714 (2018) 21 December 2018
SCIENCE IMMUNOLOGY
|
RESEARCH ARTICLE
2 of 19
no more than 1/600,000 individuals worldwide. Here, we tested the
hypothesis that two common and catalytically inactive missense
TYK2 variants, P1104A and I684S (39), might underlie MSMD,
tuberculosis, or both.
RESULTS
Ten homozygotes for TYK2 P1104A suffered from
mycobacterial diseases
The common TYK2 variants P1104A (rs34536443) and I684S
(rs12720356) are both catalytically impaired, as shown by in vitro
kinase assays in reconstituted TYK2-deficient fibrosarcoma cells
(U1A cells) (39). Other studies with selective small-molecule kinase
inhibitors suggested that the catalytic activity of TYK2 was required
for T cell responses to IL-12 and IL-23, but not IFN- and IL-10
(40). Consistently, the P1104A variant has been reported to im-
pair cellular responses to both IL-12 and IL-23 in human memory
T cells, whereas discordant results were obtained for IFN- (39, 41).
The response to IL-10 was normal in human leukocytes (41). On
the basis of the gnomAD database (42) (gnomAD: http://gnomad.
broadinstitute.org), these two missense variants are rare (<0.02%)
in East Asian populations, but otherwise common (>0.8%) in the
other four main gnomAD populations, reaching their highest fre-
quencies in Europeans (4.2% for P1104A and 9% for I684S) (fig. S1,
A and B) (43, 44). On the basis of the 1000 Genomes Project data-
base (45), these two variants are not in linkage disequilibrium.
We investigated the possibility that these variants might confer a
predisposition to MSMD, tuberculosis, or both. We screened our
whole- exome sequencing (WES) data for 463 patients with MSMD
and 291 children with tuberculosis, from different geographic loca-
tions and ancestries, and for 163 adults of North African ancestry
with early-onset pulmonary tuberculosis (table S1). None of these
patients carried pathogenic mutations in known MSMD- and
tuberculosis-
causing genes (12, 46). Our WES data for 2835 other
patients, from various ethnic origins (fig. S1C) and with various
genetically unexplained non-mycobacterial infections, were used as
a control. Among the 3752 exomes available in total, we identified
366 I684S heterozygotes, 168 P1104A heterozygotes, 18 I684S
homozygotes, and 6 I684S/P1104A compound heterozygotes, with
no clustering of any of these genotypes within any of the patient
cohorts (table S1). By contrast, we identified 11 unrelated P1104A
homozygotes, which were confirmed by Sanger sequencing: 7 with
tuberculosis (3 children under the age of 15 years and 4 adults
under the age of 40 years), 3 with MSMD (all under 3 years of age),
and 1 with CMC (aged 1 year) (Fig.1, A to C; fig. S1D; and Supple-
mentary Materials and Methods). We further Sanger sequenced
TYK2 in parents and siblings of these 11 patients. We found that, in
kindred K with the CMC patient, homozygosity for P1104A did
not segregate with CMC, because one sibling with CMC was het-
erozygous for P1104A, implying that there is another genetic cause
for CMC in this kindred (fig. S1D). We also found only one as-
ymptomatic P1104A homozygote among the relatives of the other
10 patients (kindred G, I.1). In total, we identified 10 unrelated P1104A
TYK2 homozygotes with MSMD (3 patients) or primary tuberculosis
(7 patients).
P1104A homozygosity is strongly enriched in patients
with tuberculosis
Principal components analysis (PCA) based on the WES data
(fig. S1C) (47) confirmed the diverse ancestries of the 10 patients.
Eight were living in their countries of origin (Fig.1C and fig. S1B).
The Mexican patient was living in the United States, and the 10th
patient, who was living in Brazil, had mixed European and African
ancestry. We compared the proportions of individuals with P1104A
in each cohort and estimated odds ratios (ORs) by logistic regres-
sion, with adjustment for the first three principal components of the
PCA to account for ethnic heterogeneity (48). In addition to the
2835 exomes already used as controls, we used all 2504 available
1
St. Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller Branch, Rockefeller University, New York, NY, USA.
2
Laboratory of Human Genetics of Infectious
Diseases, Necker Branch, INSERM U1163, Paris, France.
3
Paris Descartes University, Imagine Institute, Paris, France.
4
Cytokine Signaling Unit, Pasteur Institute, Paris, France.
5
INSERM U1221, Paris, France.
6
Human Evolutionary Genetics Unit, Pasteur Institute, Paris, France.
7
CNRS UMR2000, Paris, France.
8
Center of Bioinformatics, Biostatistics
and Integrative Biology, Pasteur Institute, Paris, France.
9
Immunology Division, Garvan Institute of Medical Research, Darlinghurst, New South Wales, Australia.
10
Division
of Clinical Immunology, Department of Laboratory Medicine, Karolinska Institute, Karolinska University Hospital Huddinge, Stockholm, Sweden.
11
Department of Clinical
Translational Research, Singapore General Hospital, Singapore, Singapore.
12
Department of Biomedical and Health Sciences, University of Vermont, Burlington, VT, USA.
13
St. Vincent's Clinical School, University of New South Wales, Darlinghurst, New South Wales, Australia.
14
Sidra Medicine, Doha, Qatar.
15
Department of Biochemistry,
University of Montreal, Montreal, Quebec, Canada.
16
Department of Biochemistry, Microbiology, and Immunology, University of Ottawa, Ottawa, Ontario, Canada.
17
Im-
munology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA.
18
Institute of Biochemical Sciences, Faculty of Medicine, Uni-
versity of Chile, Santiago, Chile.
19
Laboratory of Microbiology, Clinical Laboratory Department School of Medicine, Pontifical Catholic University of Chile, Santiago, Chile.
20
The Charles Bronfman Institute for Personalized Medicine, Icahn School of Medicine at Mount Sinai, New York, NY, USA.
21
Department of Genetics and Genomic Sciences,
Icahn School of Medicine at Mount Sinai, New York, NY, USA.
22
Department of Pediatrics, Memorial Sloan Kettering Cancer Center, New York, NY, USA.
23
Department of
Pediatric Pulmonology, Necmettin Erbakan University, Meram Medical Faculty, Konya, Turkey.
24
Meram Faculty of Medicine, Department of Internal Medicine, Division of
Allergy and Immunology, Necmettin Erbakan University, Konya, Turkey.
25
Jeffrey Modell Center for Diagnosis and Research in Primary Immunodeficiencies, Faculty of
Medicine University of La Frontera, Temuco, Chile.
26
Department of Pediatrics, Federal University of São Paulo Medical School, São Paulo, Brazil.
27
Department of Immu-
nology, Institute of Biomedical Sciences, and Institute of Tropical Medicine, University of São Paulo, São Paulo, Brazil.
28
National Institute of Genomic Medicine, Mexico
City, Mexico.
29
Department of Molecular Biology and Genetics, Bilkent University, Ankara, Turkey.
30
Department of Pneumology, Military Hospital Mohammed V, Rabat,
Morocco.
31
Institute of Clinical and Molecular Virology, University of Erlangen-Nuremberg, Erlangen, Germany.
32
Research Center for Immunodeficiencies, Pediatrics
Center of Excellence, Children's Medical Center, Tehran University of Medical Sciences, Tehran, Iran.
33
Department of Infectious Diseases, Medical School, Pontifical Catholic
University of Chile, Santiago, Chile.
34
Department of Pediatrics, San Sebastián University, Santiago, Chile.
35
Department of Infectious Diseases and Pediatric Immunology,
School of Medicine, Pontifical Catholic University of Chile, Santiago, Chile.
36
Department of Medicine, Immunobiology Program, University of Vermont, Burlington, VT,
USA.
37
Seattle Children's Research Institute and Department of Pediatrics, University of Washington, Seattle, WA, USA.
38
Department of Pediatric Immunology and
Allergy, Necmettin Erbakan University, Meram Medical Faculty, Konya, Turkey.
39
Genetics Unit, Military Hospital Mohamed V, Hay Riad, Rabat, Morocco.
40
Center for the
Study of Primary Immunodeficiencies, AP-HP, Necker Hospital for Sick Children, Paris, France.
41
Beijing Genomics Institute BGI-Shenzhen, Shenzhen, China.
42
Pediatric
Hematology-Immunology Unit, Necker Hospital for Sick Children, AP-HP, Paris, France.
43
Howard Hughes Medical Institute, New York, NY, USA.
*Corresponding author. Email: stbo603@rockefeller.edu (S.B.-D.); jean-laurent.casanova@rockefeller.edu (J.-L.C.)
†These authors contributed equally to this work.
‡These authors contributed equally to this work.
§These authors contributed equally to this work.
||These authors contributed equally to this work.
Boisson-Dupuis et al., Sci. Immunol. 3, eaau8714 (2018) 21 December 2018
SCIENCE IMMUNOLOGY
|
RESEARCH ARTICLE
3 of 19
Kindred A Kindred D
P2
1 2
12
Kindred E
m/m
1
WT/m
2
1
WT/m
Kindred I
m/m
P1
1
E?
2
1
E?
Kindred F
m/m
P4
1
E?
2
1
E?
Kindred G
m/m
P5
1
WT/m
2
12
m/m
Kindred C
E? m/m
P7
1
WT/m
2
12
Kindred H
E? m/m
P8
E?
E? E?
1
E?
2
1
E?
m/m
P3
WT/WT
WT/m WT/m
1
E?
2
1
E?
m/m
P6
Kindred B
3 4
1
E?
2
1
E?
m/m
P9
WT/m
3
FERM
SH2 Pseudokinase
Kinase
NH2 COOH
C70HfsX21
L767X
T1106HfsX4
E154XS50HfsX1 R638X
P1104A
P216HfsX14
I684S
I
II
3
I
II
MSM
D
TB
MSMD
TB
A
B
C
D
E
WT/m
451584 8891187
28
1567 8751173
0.1 0.2 0.3 0.4 0.50.0 0.1 0.2 0.3 0.4 0.50.0
0.1 0.2 0.3 0.4 0.50.0
0 500 1000 1500
0 500 1000 1500
0 500 1000 1500
I684S TYK2 M694V MEFV C282Y HFE
Controls
(n = 5339)
Homoz. Homoz. OR Homoz. OR Homoz. OR
carriers carriers (95% CI) carriers (95%CI)carriers(95% CI)
89.3123.53 53.72
(14.7–1725) (2.9–483)(10.1–993)
0.46 0.76 0.61
(0.03–2.2)(0.12–2.6)(0.15–1.8)
10 4.87 × 10
−8
I684S221 0.38 20.713 0.4
Variant P value P value P value
P1104A 17 8.37 × 10
−8
33.27 × 10
−3
Tuberculosis
(n = 454)
MSMD
(n = 463)
TB + MSMD
(n = 917)
Current frequency in western Europe
0
0.1 0.2 0.3 0.4 0.5
500 1000 1500 2000
0.0
Number of variants
P1104A TYK2
Kindred J
1
E?
2
1
E?
m/m
P10
CountryMAF TB incidence* BCG Age of onset of
Patients Disease
of residence
Origin by PCA
gnomAD#(/100,000) vaccination symptoms (years)
P1 BCG osteomyelitis Sweden European 0.0429.2 Yes1
P2 MAC osteomyelitis USA American/Mexican 0.0123.2 No 1
P3 BCG disseminated IranMiddle Eastern0.031 15 Yes2
P4 PulmonaryBrazilMixed European/African 0.01841Yes 6
P5 PulmonaryAlgeria North African 0.01875Yes 40
P6 Pulmonary Morocco North African 0.018107 Yes27
P7 Miliary Turkey Turkish0.021 18 Yes15
P8 Pulmonary Chile American/Chilean 0.01216Yes 13
P9 Pulmonary Morocco North African 0.018107 Yes35
P10Pulmonary Chile American/Chilean 0.01216Yes 33
#: Allele frequency in the country of origin in the gnomAD database
*TB incidence in the country of residence from WHO 2015
%
Homoz@
0.41
1.58
3.27
2.85
1.48
4.16
5.53
0.6
0.82
0.27
@: Percentage of homozygosity
TYK2
Fig. 1. Familial segregation and
clinical information for patients
homozygous for TYK2 P1104A.
(A) Schematic diagram of the TYK2
protein with its various domains
(FERM, SH2, pseudokinase, and
tyrosine kinase). The positions of
the previously reported TYK2 mu-
tations resulting in premature STOP
codons are indicated in red. The
positions of the I684S and P1104A
polymorphisms are indicated in blue
and green, respectively. (B) Pedi-
grees of the 10 TYK2-deficient
families. Each generation is desig-
nated by a Roman numeral (I–II),
and each individual by an Arabic
numeral. The double lines connect-
ing the parents indicate consan-
guinity based on interview and/
or a homozygosity rate of >4%
estimated from the exome data.
Solid shapes indicate disease status.
Individuals whose genetic status
could not be determined are in-
dicated by “E?”, and “m” indicates
a TYK2 P1104A allele. (C) Sum-
mary table of clinical details and
origin of the patients associated
with the MAF in the country of ori-
gin. The incidence of tuberculosis
(TB) in the country of residence is
also mentioned. MAC indicates
Mycobacterium avium complex.
(D) Summary of WES, indicating
the numbers of individuals with
tuberculosis or MSMD and of con-
trols carrying the I684S or P1104A
variant of TYK2 in the homozygous
state, and the associated P value
and OR. (E) Distributions of the cur-
rent allele frequencies of variants
that segregated 4000 years ago at
frequencies similar to those of the
P1104A and I684S TYK2, M694V
MEFV, and C282Y HFE variants.
The red vertical lines indicate the
current frequency of the four
variants of interest. Colored bars
indicate the distribution of cur-
rent allele frequency, in the 1000
Genomes Project, for variants with
frequencies in ancient European
human DNA similar to those of
the four candidate variants (52).
Black lines indicate the distribu-
tion of simulated frequencies, in
the present generation, for alleles
with a past frequency similar to
that of the four candidate variants,
with propagation over 160 gen-
erations (corresponding to a pe-
riod of ~4000 years) under the Wright-Fisher neutral model. For instance, for the P1104A allele, which had a frequency of ~9% in ancient Europeans, colored bars
indicate the observed distribution of current frequencies for the 31,276 variants with a frequency of 8 to 10% 4000 years ago. The black lines indicate the distribution
of frequencies for 100,000 simulated alleles obtained after 160 generations under the Wright-Fisher neutral model.
Boisson-Dupuis et al., Sci. Immunol. 3, eaau8714 (2018) 21 December 2018
SCIENCE IMMUNOLOGY
|
RESEARCH ARTICLE
4 of 19
individuals from the 1000 Genomes Project (45), giving a total of
5339 controls for whom we have complete WES data (Fig.1D).
P1104A homozygosity was more enriched among patients with
MSMD than among controls [P = 3.27 × 10
−3
; OR, 23.53; 95%
confidence interval (CI), 2.9 to 483], and an even higher level of
enrichment was observed among patients with tuberculosis (P =
8.37 × 10
−8
; OR, 89.31; 95% CI, 14.7 to 1725). The level of enrich-
ment in homozygosity for this variant was intermediate but more
significant when both groups were analyzed together (OR, 53.72;
95% CI, 10.1 to 993; P = 4.87 × 10
−8
). By contrast, no enrichment
in homozygosity for this variant was observed among the patients
with other infections studied in the laboratory (table S1) (49, 50).
Aside from the 10 MSMD and tuberculosis patients, we identi-
fied only one other P1104A homozygote by WES: a CMC patient
whose P1104A homozygosity was not CMC-causing, living in the
United States, where infants are not inoculated with BCG and
M. tuberculosis is not endemic (fig. S1D). No homozygotes were
observed among the 2504 individuals of the 1000 Genomes Project.
No significant enrichment in P1104A heterozygosity was observed
in any of the cohorts studied, including patients with MSMD (P =
0.57) or tuberculosis (P = 0.49), demonstrating the recessive na-
ture of P1104A inheritance for both mycobacterial conditions.
Moreover, no significant enrichment in I684S heterozygotes or
homozygotes or in P1104A/I684S compound heterozygotes was
observed in any of the cohorts studied (table S1). Last, the TYK2
P1104A allele yielded the highest OR at genome-wide level in an
independent enrichment analysis performed under the assump-
tion of a recessive mode of inheritance and considering all com-
mon missense or potential loss-of-function (LOF) alleles in our
entire cohort of 3752 patients (fig. S1E). These results strongly sug-
gest that homozygosity for P1104A is a genetic etiology of primary
tuberculosis and MSMD.
TYK2 P1104A allele frequency has decreased in Europe over
the past 4000 years
The higher risk of life-threatening tuberculosis in P1104A homo-
zygotes suggests that this variant has been subject to negative
selection in areas in which this disease has long been endemic, such
as Europe (51). We analyzed changes in the frequencies of the
P1104A and I684S TYK2 variants in the European population,
from ancient to modern times (52). Only three nonsynonymous
TYK2 variants—P1104A, I684S, and V362F—were found in an
available sample of central European individuals who lived during
the late Neolithic age ~4000 years ago (52). Over this period, the
frequency of TYK2 P1104A has significantly decreased in Europeans,
from about 9% to 4.2% (Fig.1E). Of the 31,276 variants with fre-
quencies in the 8 to 10% range 4000 years ago, P1104A is among
the 5% displaying the largest decrease in frequency (empirical P =
0.048; Fig.1E). Furthermore, the neutral model of evolution was
significantly rejected for P1104A in Wright-Fisher simulations
(simulation P = 0.050; Fig.1E and Supplementary Materials and
Methods), suggesting an absence of bias in the empirical analyses.
As a negative control, the frequency of V362F remained stable
(from 25% to 26.2%) and that of I684S did not decrease signifi-
cantly over this period (empirical P = 0.181). The frequency of
I684S was about 14% 4000 years ago and is now 9%, placing this
variant among the 80% of the 36,469 polymorphisms considered
with a frequency that was in the 13 to 15% range 4000 years ago and
has remained relatively stable.
TYK2 P1104A allele was possibly purged in Europe
by tuberculosis
We subsequently analyzed, as positive controls, two relatively com-
mon mutations known to cause life-threatening AR disorders and
present in ancient Europeans: the MEFV M694V variant underly-
ing Mediterranean fever (MF) (53) and the HFE C282Y underlying
hemochromatosis (which also decreases male fertility) (54). Both
these variants decreased significantly in frequency over the same
period, from about 11% to 0.4% for MEFV M694V and from 16% to
5.7% for HFE C282Y (empirical P = 0.016 for both variants; Fig.1E).
Therefore, our preliminary assessments suggest that TYK2 P1104A,
MEFV M694V, and HFE C282Y have been subject to negative
selection in Europeans, whereas TYK2 I684S has not. The stronger
selection operating on MEFV M694V, and to a lesser extent HFE
C282Y, than on TYK2 P1104A is consistent with the inevitability of
MF and hemochromatosis in patients with these mutations, whereas
tuberculosis development also requires exposure to M. tuberculosis.
These results suggest that, unlike I684S, P1104A has been undergoing
a purge in Europe since the Neolithic period due to the continued
endemic nature of life-threatening tuberculosis (51). No other in-
tramacrophagic infection, whose control depends on IFN-, has
been endemic for so long in Europe (55, 56). The purging of delete-
rious mutations is expected to be much less effective in the absence
of continued exposure (57, 58), which has been the case for other
infections that killed a sizeable proportion of Europeans, albeit for
no more than several decades or a few centuries, such as plague
(59). The observed decline in P1104A allele frequency is consistent
with the purging of a recessive trait that kills in childhood or when
the individual is of reproductive age. This decrease would be much
steeper for a dominant trait with a similar fitness effect. These re-
sults suggest that homozygosity for P1104A, which is still present in
about 1/600 Europeans and between 1/10,000 and 1/1000 individuals
in other regions of the world, with the exception of East Asia, where the
allele is almost absent, has been a major human genetic determinant
of primary tuberculosis during the course of human history.
TYK2 P1104A impairs IL-23 but not IFN-, IL-12,
and IL-10 signaling
We performed a functional characterization of the I684S and
P1104A TYK2 alleles, focusing on the four known human TYK2-
dependent signaling pathways (10). In reconstituted U1A cells
stimulated with IFN- in vitro, both mutant proteins were previ-
ously shown to be catalytically inactive, i.e., unable to autophos-
phorylate or phosphorylate a substrate such as signal transducer
and activator of transcription 3 (STAT3) (39). However, both could
be phosphorylated by Janus kinase 1 (JAK1), unlike the prototypical
kinase-dead adenosine 5′ triphosphate (ATP)–binding mutant K930R
(39). Epstein-Barr virus (EBV)–transformed B (EBV-B) cells and her-
pesvirus saimiri (HVS)–transformed T (HVS-T) cells derived from
a TYK2-deficient patient without TYK2 protein expression (10) were
stably transduced with a retrovirus generated with an empty vector
or a vector containing the wild-type (WT), P1104A, I684S, or
K930R TYK2 complementary DNA (cDNA) (60). Transduction
with the WT or any mutant TYK2 restored both TYK2 expres-
sion, as shown by Western blotting, and the corresponding TYK2
scaffolding-dependent surface expression of IFN-R1, IL-10R2,
and IL-12R1, as shown by flow cytometry (Fig.2, A and B, and
fig. S2, A and B). In P1104A-expressing cells, the IFN-– and IL-12–
dependent signaling pathways were normal, as shown by the levels
Boisson-Dupuis et al., Sci. Immunol. 3, eaau8714 (2018) 21 December 2018
SCIENCE IMMUNOLOGY
|
RESEARCH ARTICLE
5 of 19
AB
CD
E
TYK2
GAPDH
EV
EV
I684S
P1104A
WT
K930R
I684S
P1004A
WT
K930R
TYK2
−/−
EBV-B cells TYK2
−/−
HVS-T cells
EV
WT
P1104A
I684S
K930R
0
500
1000
1500
2000
2500
MFI
***
EV
WT
P1104A
I684S
K930R
0
1000
2000
3000
4000
***
EV
WT
P1104A
I684S
K930R
0
2000
4000
6000
TYK2
−/−
EBV-B cells TYK2
−/−
HVS-T cells
EV
I684S
P1104A
WT
K930R
+−+−+−+
−+
−
IL-23
pTYK2
pJAK2
TYK2
JAK2
GAPDH
TYK2
−/−
EBV-B cells
EV
I684S
P1104A
WT
K930R
+−+−+−+−+−
pTYK2
pJAK1
TYK2
JAK1
GAPDH
TYK2
−/−
EBV-B cells
EV
I684S
P1104A
WT
K930R
+−+−+−+−+−
IL-12
pTYK2
pJAK2
TYK2
JAK2
GAPDH
TYK2
−/−
HVS-T cells
pSTAT1
STAT1
TYK2
GAPDH
pSTAT4
STAT4
TYK2
G
F
pSTAT3
150
150
38
150
150
150
38
150
150
38
150
150
150
150
38
150
150
MW
MW MW
MW
102
102
38
150
102
102
76
76
150
TYK2
Tubulin
52
pSTAT1
STAT1
STAT3
TYK2
−/−
HVS-T cells
102
102
150
MFI pSTAT4
0
200
400
600
800
1000
−−−+ +− −− −+
EV
WT
P1104
A
I684
S
K930R
IL-12
+−−− −+ +− −−
+− −
−+ −
+−
−+
**
***
ns
**
***
***
***
ns
ns
ns
102
STAT1
102
pSTAT1
GAPDH
38
EV
WT
P1101A
I681S
E779K
0.00
0.05
0.10
0.15
0.20
Mouse TYK2
−/−
MEF cells and VSV
Human TYK2
−/−
U1A cells and VSV
*
ns
*
*
*
ns
ns
EV
WT
P1104A
I684S
0.00
0.02
0.04
0.06
0.08
0.10
***
Fig. 2. Cellular responses to IFN-, IL-12 and IL-23 in transduced EBV-B and HVS-T cells. TYK2-deficient EBV-B and HVS-T cells were transduced with a retrovirus gener-
ated with an empty vector (EV), or vectors encoding WT TYK2, or the P1104A, I684S, or K930R TYK2 alleles. (A) Levels of TYK2 in transduced EBV-B (left) and HVS-T (right)
cells, as determined by Western blotting. (B) Levels of IL-12R1 and IFN-R1 in transduced EBV-B (left) and HVS-T (right) cells, as determined by flow cytometry. ***P <
0.001, two-tailed Student’s t test. Error bars indicate SEM. (C, D, and F) Phosphorylation of JAKs and STATs in unstimulated (−) transduced EBV-B or HVS-T cells or in these cells
after stimulation (+) with IFN- (C) (pTYK2, pJAK1, and pSTAT1), IL-12 (D) (pTYK2, pJAK2, pSTAT1, and pSTAT4), and IL-23 (F) (pTYK2, pJAK2, pSTAT3, and pSTAT1), as assessed
by Western blotting with specific antibodies recognizing phospho-TYK2, phospho-JAK1, phospho-JAK2, phospho-STAT1, phospho-STAT4, and phospho-STAT3. MW,
molecular weight. (E) Phosphorylation of STAT4 in response to IFN- and IL-12, as determined by flow cytometry in HVS-transduced T cells and expression as mean fluorescence
intensity (MFI). **P < 0.01, ***P < 0.001, two-tailed Student’s t test. ns, not significant. (G) IFN- response of U1A (left) and MEF (right) cells, both lacking TYK2, after transduc-
tion with the indicated human and mouse TYK2 alleles, respectively, or with empty vector control, as measured in an IFN-–induced antiviral activity assay (see Materials
and Methods). A unique dose is shown: an IFN- dose of 0.01 ng/ml for human cells and 1 IU/ml for mouse cells.
