Homozygosity for the catalytically inactive P1104A missense variant of the TYK2 Janus kinase selectively disrupts the induction of IFN-γ by IL-23 and is a common monogenic etiology of tuberculosis.
Abstract:
Inherited IL-12Rβ1 and TYK2 deficiencies impair both IL-12- and IL-23-dependent IFN-γ 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 individuals 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-α, IL-10, or even IL-12, which, like IL-23, induces IFN-γ 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-γ by IL-23 and is a common monogenic etiology of tuberculosis.
#: 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
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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
(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
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RESEARCH ARTICLE
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AB
CD
E
TYK2
GAPDH
EV
EV
I684S
P1104A
WT
K930R
I684S
P1004A
WT
K930R
TYK2
−/−
EBV-B cellsTYK2
−/−
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 cellsTYK2
−/−
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
MWMW
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.
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Q1. What are the contributions in "Tuberculosis and impaired il-23–dependent ifn- immunity in humans homozygous for a common tyk2 missense variant" ?
For example, Boisson-Dupuis et al. this paper showed that the IL-12R1 deficiency in primary tuberculosis patients can be linked to weakly virulent mycobacteria, such as the Bacille Calmette-Guérin vaccine and environmental species.
Q2. What is the reason for the impairment of IFN production in P1104A homozygo?
T cells of P1104A homozygotes have impaired IFN- production, due to their very weak response to IL-23, accounting for the susceptibility to MSMD or primary tuberculosis.
Q3. What is the role of TYK2 in antimycobacterial immunity?
the TYK2-dependent response to IL-23 that is disrupted by P1104A is essential for antimycobacterial IFN- immunity, but seems to be redundant for anti-fungal IL-17 immunity, given the absence of Candida infection in the patients described here.
Q4. What is the effect of TYK2 variants on cellular responses to IL-12?
The impact of TYK2 variants on cellular responses to IL-12 and IL-23 is irrelevant in nonhematopoietic cells, because the receptors for these cytokines are expressed only on leukocytes.
Q5. What is the CI for the TYK2 P1104A homozygos?
Several genome-wide association studies have shown that homozygosity for TYK2 P1104A has a strong protective effect (ORs ranging from 0.1 to 0.3) against various autoinflammatory or autoimmune conditions (41).
Q6. What grants were awarded to the Laboratory of Human Genetics of Infectious Diseases?
Funding: The Laboratory of Human Genetics of Infectious Diseases was supported, in part, by grants from the French National Agency for Research (ANR) under the “Investissement d’avenir” program (grant no. ANR-10-IAHU-01), the TBPATHGEN project (grant no.
Q7. What is the effect of negative selection on TYK2 P1104A?
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.
Q8. How did the authors determine the frequency of TYK2 variants in Europeans?
The authors analyzed the occurrence of negative selection acting on the two TYK2 variants by testing whether their frequency in Europeans has decreased more than other variants that were in the same frequency range 4000 years ago.
Q9. What is the common etiology of tuberculosis in Europe?
Between 1/10,000 and 1/1000 individuals are homozygous in endemic regions of the world (other than East Asia), where P1104A TYK2 is likely to define a strictly recessive but relatively common etiology of severe primary tuberculosis (about 0.5% of cases).
Q10. What was the phenotype of TYK2 in P1104A homozy?
In P1104A homozygous cells, the response to IFN- was modestly reduced in terms of JAK1, TYK2, STAT3, and STAT1 phosphorylation (Fig. 3C and fig.
Q11. What is the level of enrichment in homozygosity?
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).