A Novel Homozygous Mutation Causing Complete TYK2 Deficiency, with Severe Respiratory Viral Infections, EBV-Driven Lymphoma, and Jamestown Canyon Viral Encephalitis

Autosomal recessive tyrosine kinase 2 (TYK2) deficiency is characterized by susceptibility to mycobacterial and viral infections. Here, we report a 4-year-old female with severe respiratory viral infections, EBV-driven Burkitt-like lymphoma, and infection with the neurotropic Jamestown Canyon virus. A novel, homozygous c.745C > T (p.R249*) variant was found in TYK2. The deleterious effects of the TYK2 lesion were confirmed by immunoblotting; by evaluating functional responses to IFN-α/β, IL-10, and IL-23; and by assessing its scaffolding effect on the cell surface expression of cytokine receptor subunits. The effects of the mutation could not be pharmacologically circumvented in vitro, suggesting that alternative modalities, such as hematopoietic stem cell transplantation or gene therapy, may be needed. We characterize the first patient from Canada with a novel homozygous mutation in TYK2.


Introduction
Tyrosine kinase 2 (TYK2) is a member of the Janus kinase (JAK) family of kinases that, together with signal transducer and activator of transcription (STAT) molecules, constitute the JAK-STAT pathway, transducing signals downstream of multiple cytokine receptors to mediate innate and adaptive immune responses.
Herein, we characterize a novel p.R249* mutation in TYK2 from a child with numerous life-threatening viral infections, while expanding the spectrum to include the neurotropic Jamestown Canyon virus.

Subjects
Subjects and the patient's family members provided informed consent on McGill University Health Centre (MUHC) institutional review board-approved research protocol (GEN10-256).Comprehensive medical histories, including review of all available outside records and serial clinical evaluations at the local institution, as well as immunologic laboratory testing at the MUHC, were performed.

Sequencing and Bioinformatics Analysis
Whole-exome sequencing (WES) was performed at a CLIAcertified diagnostic laboratory (GeneDx, Gaithersburg, MD, US).For Sanger sequencing, the TYK2 gene was PCR amplified from genomic DNA using primers designed to flank the respective regions (primers and sequencing conditions available on request).Sequencing was performed at the Génome Québec Innovation Centre.Sequencing analyses were performed on the Sequencher® sequence analysis software (Gene Codes Corporation, Ann Arbor, MI).

DNA Constructs and Transfection
The myc-flag tagged wild-type (WT) TYK2 plasmid was purchased from OriGene and confirmed WT by Sanger sequencing.The WT TYK2 plasmid was used to create the TYK2 c.745C > T variant using site-directed mutagenesis kit (New England Biolabs).Liposomal transfection in A549 cells was done with the Lipofectamine 3000 Transfection Reagent (Invitrogen) according to the manufacturer's instructions.Cells were harvested 48 h after transfection.

Immunoblotting
LCL or A549 (obtained by trypsinization) were resuspended in RIPA lysis buffer (Sigma) supplemented with cOmplete Mini Protease Inhibitor Cocktail (Roche) and PhosSTOP (Roche), then boiled in LDS sample buffer and Blot Sample Reducing Agent (Thermo Fisher Scientific).Protein was separated by SDS-PAGE on premade gels (Novex) before transfer to the membrane via the iBlot Gel Transfer Device (Invitrogen).The membrane was blocked in 5% skim milk TBST to block nonspecific protein binding.The membrane was blotted using primary antibodies overnight at 4 °C.The membrane was washed 4 times with 0.1% Tween 20 in TBS and then incubated with a 1:15,000 dilution of secondary antibodies in blocking buffer.The membranes were scanned and analyzed with an Odyssey IR scanner using Odyssey imaging software 3.0 (LI-COR Biosciences, Inc).

Quantitative Real-Time PCR
RNA was extracted using TRIzol Reagent (Invitrogen) according to the manufacturer's instructions and quantified by NanoDrop-100 spectrophotometer; 500 ng of RNA was reverse transcribed using the Maxima H Minus First Strand cDNA Synthesis Kit (Thermo Fisher Scientific).Realtime quantitative PCR was performed with CFX Opus 96 Real-Time PCR (Bio-Rad Laboratories).TaqMan quantitative PCR assays were run using primers/probes: CXCL11 (Hs00171138), IRF1 (Hs00971965), MX1 (Hs00895608), OAS2 (Hs00942643), ISG15 (Hs00192713), IL-8 (Hs00174103), and GAPDH (Hs02758991).The mRNA input was normalized to the expression of the housekeeping genes, GAPDH [8].Statistical significance of the data was validated using one-way ANOVA with Tukey's post hoc test and multiple comparison procedure.The level of significance was set at *p < 0.05.

Flow Cytometry
Cells were blocked in Fc receptor binding inhibitor monoclonal antibody (Invitrogen) and incubated with LIVE/ DEAD Fixable Dead Cell Stain (Thermo Fisher Scientific).For intracellular staining, cells were stained using the FIX & PERM Cell Fixation and Cell Permeabilization Kit (Thermo Fisher Scientific).Samples were analyzed using 4-laser Cytek Aurora flow cytometer (Cytek Biosciences).Data was done using FlowJo (Ashland, OR).For statistical analysis, repeated-measures one-way ANOVA, followed by Tukey's multiple comparison post-test, was used.The level of significance was set at *p < 0.05.

Case Report
Patient 1 (P1) is a 4-year-old female born to parents of Pakistani origin with distant consanguinity (see Supplemental Appendix for complete clinical details).At 6 weeks of age, she developed respiratory failure due to influenza B, from which she recovered.Over the next few months, she developed recurrent respiratory infections requiring supplemental oxygen and treatment with bronchodilators and systemic corticosteroids.At 8 months of age, she was diagnosed with bilateral subdural hygromas, which eventually resolved.At 11 months old, she developed severe respiratory disease due to Enterovirus/Rhinovirus requiring hospitalization and oxygen supplementation, but not mechanical ventilation, from which she recovered.She received all routine immunizations, including live viral vaccines, without adverse events; she never received BCG vaccination.At age 4 years, she developed a rapidly growing neck mass that invaded the left sternocleidomastoid, mediastinum, and retroperitoneum.A mass was also noted in the recto-uterine pouch.Analysis of the mass revealed Burkitt lymphoma, with tumor cells positive for EBER; bacterial, mycobacterial, and fungal cultures yielded no organism.She was treated with R-COPADM-1 protocol (rituximab, vincristine, prednisone, methotrexate, folinic acid, cyclophosphamide, doxorubicin, cytarabine) followed by R-CYM2 (rituximab, methotrexate, folinic acid, cytarabine).During her active chemotherapy, she developed multiple viral infections including Coronavirus OC43/HKU1, rotavirus and norovirus gastroenteritis, and enterovirus/rhinovirus URTI.In the context of screening for high-risk exposure, she was found to be PCR-positive for SARS-CoV-2 (B.1.1.529VOC; Omicron); she manifested only with coryza.Five weeks later, her PCR test reverted to negative.Two weeks later, though, she had new cough and fever and was SARS-CoV-2 PCR-positive again; she did not require oxygen supplementation and resolved without complications.Three months after successful completion of her chemotherapy, she developed a febrile, tonic-clonic seizure.Imaging revealed focal areas of T2/T2 FLAIR hyperintensity and edema, with corresponding restricted diffusion in the cortex of bilateral parieto-occipital and posterior temporal lobes.Cerebrospinal fluid PCR analysis yielded Jamestown Canyon virus; Jamestown Canyon serology was IgM-positive by ELISA and 1:40 by plaque reduction neutralization test.She clinically stabilized but was left with significant neurologic impairment.Given her history and fulminant presentation, further investigations were performed (Immunophenotyping data, Table 1).

Homozygous p.R249* Variant in TYK2
Whole-exome sequencing (WES) in P1 identified homozygous c.745C > T (p.R249*) in TYK2 (NM_003331).This variant, which has not been previously reported, was confirmed by Sanger sequencing (Fig. 1A).Each of the parents was confirmed to carry the variant.It is predicted to cause a premature termination codon in exon 7 residing in the FERM domain (aa 28-451), which, together with the adjacent SH2-like domain, mediate its interaction with receptors (Fig. 1B); in particular, this region of TYK2 is necessary for signaling through the type I IFN receptor system [9].This variant was predicted in silico to be deleterious (e.g., SIFT, PolyPhen2; CADD Phred; MutationTaster) and is extremely rare (gnomAD allele frequency of 4.13 × 10 −6 ; no homozygotes reported).
To confirm the predicted pathogenicity of the variant, we first assessed its molecular impact on TYK2 expression by immunoblot of EBV-transformed B-lymphoblastoid cell lines (LCLs) from P1 vs. health controls [HC], showing a loss of expression in P1 (Fig. 1C).We confirm this result in an autonomous A549 cell model system in which endogenous TYK2 was knocked out by CRISPR-Cas9-based technology, then transiently transfected with either wildtype (WT) TYK2 allele or the c.745C > T variant (TYK2 p.R249*) (Fig. 1D).In this latter over-expression system, immunoblotting against TYK2 WT demonstrates a smear of isoforms, most likely related to the known post-translational modifications (e.g., ubiquitination; phosphorylation) of TYK2 at multiple residues, affecting its stability/ decay [10].However, no TYK2 protein was detected from the c.745C > T allele, by immunoblotting against the TYK2 protein or against the C-terminal-myc-tag, confirming its loss of expression.

Kinase Activity of TYK2 p.R249* is Impaired in P1's LCL
We first analyzed the capacity for auto-and trans-phosphorylation of TYK2 by immunoblotting in P1 and HC LCLs following stimulation with either IFNα/β or IL-10.In contrast to responses seen in HC, the absence of TYK2 expression, as well as its phosphorylation, were observed in P1's LCLs (Fig. 2A).
We then assessed the impact of TYK2 R249* on its downstream kinase activity within the JAK/STAT canonical signaling pathway, by flow cytometry-based measurement of phosphorylation of the respective STAT proteins that mediate signaling from IFNα/β, IL-10, and IL-23 (Fig. 2B).For all tested stimuli, P1 LCLs show reduced STAT activation relative to controls, consistent with its lack of TYK2 protein expression.To confirm these anomalies in a patient-independent cell model, an A549 cell line with TYK2 knocked out was then complemented with either empty vector (EV), TYK2 WT, or p.R249* construct through transient transfection.No increase in phosphorylated STAT1 or STAT2 via IFNα/β stimulation was observed in A549 cells with either EV or bearing the p.R249* allele.
We further evaluated the p38-mediated non-canonical signaling pathway for type I IFN by assessing phosphorylation of p38 in A549 cell lines, given the high basal phospho-p38 (p-p38) activity in LCL.In the absence of TYK2 or in the presence of TYK2 R249* , the phosphorylation of p38 is very low in response to IFN stimulation (Fig. 2D).
These results indicate that the identified c.745C > T variant in TYK2 results in complete loss of all its known kinase activity in various human cytokine signaling pathways.

Scaffolding Function of TYK2 on the Expression of Associated Cytokine Receptors
The FERM and SH2 domains of TYK2 also possess scaffolding function [2,11,12], and previous patients with TYK2 deficiency have decreased cell surface expression of the cytokine receptor subunits, IFN-αR1, IL-10Rb, and IL-12Rβ1 [3].The absence of the SH2 domain and part of the FERM domain in the p.R249* mutant may similarly abolish TYK2's interaction with these molecules, thus decreasing their stability and cell surface expression.
To evaluate this, we measured the surface expression and intracellular levels of IFN-αR1, IFN-αR2, IL-10Rb, and IL-12Rβ1 on LCL by flow cytometry (Fig. 3).As previously shown, cell surface expression of IFN-αR1, IL-10Rb, and IL-12Rβ1 receptor subunits were significantly reduced in P1's LCL relative to controls (Fig. 3, left column), as were their total levels (Fig. 3, right column), confirming that the p.R249* allele causes loss of TYK2's scaffolding function.
In contrast, cell surface levels of IFN-αR2 were comparable between P1 and controls.

Attempts for Pharmacological Correctionin vitro
Given the dire clinical susceptibility to common viruses in this young child, we investigated whether the mutant TYK2 could be pharmacologically circumvented.G418, an aminoglycoside, has previously been shown to mediate ribosomal in-frame read-through of nonsense mutations [15][16][17], while 4-phenylbutyrate (4PBA) is a chemical chaperone that can rescue the expression of mutant proteins normally expressed at the plasma membrane by improving their intracellular trafficking [18][19][20].Neither agent alone, or in combination, restored TYK2 expression in P1's LCL (Supplementary figure S2).Since IFN-γ stimulation of its cognate receptor can activate JAK1 and STAT1, which are shared with the IFN-α/β signaling pathway, we sought to determine if IFN-γ may offset the diminished ISGs caused by TYK2 R249* .Of the genes tested, IFN-γ stimulation compensated some of the tunable ISGs (IRF1, IL-8), but none of the robust, antiviral ISGs (Supplementary figure S3).

Discussion
Different sub-types of TYK2 deficiency have been recently defined, based on molecular characteristics [2][3][4]: complete deficiency with no TYK2 protein production (loss of expression (LOE) and loss of function (LOF)), complete deficiency with detectable protein (residual expression but LOF), partial deficiency due to hypomorphic mutation, partial TYK2 deficiency specifically compromising IL-23 responses due to the common p.P1104A variant, and partial TYK2 deficiency specifically compromising IL-23 responses due to rare variants.Here, we present a case with a novel homozygous c.745C > T (p.R249*) mutation, leading to loss of expression and consequently, loss of kinase and scaffolding functions, resulting in complete TYK2 deficiency (LOE, LOF).
Numerous respiratory and gastrointestinal viral infections occurred in P1, including multiple episodes of lifethreatening disease, in keeping with previous reports.Intriguingly, infection with SARS-CoV-2 only resulted in non-severe/critical disease, despite not having been vaccinated against COVID-19 nor having received monoclonal or polyclonal immunoglobulin infusions.Six other pediatric patients with TYK2 deficiency who contracted SARS-CoV-2 infection have been previously reported [21]; all had complete deficiency (LOE, LOF) due to either c.647delC (p.P216Rfs*14) in 5 or homozygous c.466-1G > A in 1; four had hypoxemic COVID-19 pneumonia.The remaining two patients, along with this one reported here, may suggest that life-threatening COVID-19 shows incomplete penetrance in those with TYK2 deficiency.
On the other hand, the patient reported here developed a severe meningoencephalitis with Jamestown Canyon virus (JCV), an arthropod-borne RNA-based virus (arbovirus) belonging to the California serogroup of the Bunyaviridae family within the genus Orthobunyavirus [22].JCV is maintained in an enzootic transmission cycle involving primary reservoir and amplifying hosts (e.g., white-tailed deer) and mosquito vectors.Seroprevalence studies suggest that many, perhaps as much as 50% of JCV infections, are a-/pauci-symptomatic [23,24].Whether P1's severe JCV central nervous system (CNS) disease was due to her underlying TYK2 deficiency, previous chemotherapy, and/ or other factors is unknown at this time.However, it is notable that other CNS infections have been reported in patients with TYK2, due to herpes virus or Brucella [2], suggesting that TYK2 may underlie additional cases of severe CNS infections; further reports will help resolve this issue.
Given the centrality of TYK2 to signaling downstream from multiple receptors, we demonstrate impaired stimulation of the type I IFN receptor (IFN-I-R), IL-10R, and IL-23R, similarly to what has been previously shown [1][2][3]25].We could not test the response to IL-12 in P1 due to a lack of material, but it is expected to be impaired, like in the other TYK2-deficient patients [3].In line with previous reports of TYK2 deficient patients, and with other inborn errors of IFN-I immunity [26,27], the loss of IFN-I-R accounts for P1's unusual susceptibility to severe viral disease.Further support for this is the loss of "robust" interferon stimulated genes (ISGs), represented by expression of OAS2, ISG15 and MX1, which are primarily antiviral in action [13,14].On the other hand, "tunable" ISGs are considered to primarily arbitrate immunomodulatory and antiproliferative functions [13,14], and impaired expression of these genes may underlie the EBV-positive Burkitt lymphoma in P1 and EBV-driven lymphoproliferative disorder in other TYK2 deficiency patients [4].Interestingly, EBV encodes latent membrane protein 1 (LMP-1), an oncoprotein essential for B cell immortalization, and LMP-1 targets TYK2 [28].Although loss of IL-23 function predisposes to mycobacterial disease [29], particularly of the M. tuberculosis complex, P1 has not manifested such disease because she did not receive BCG vaccination (in keeping with provincial public health policy), and tuberculosis is not endemic where she lives.Indeed, in the largest evaluation to date, only 48% of patients with complete TYK2 deficiency had mycobacterial disease [3].Similarly, impaired IL-12R function increases susceptibility to mycobacteriosis, but also to disseminated salmonellosis or thermally dimorphic endemic mycoses [30][31][32][33][34], neither of which are prevalent in P1's region of residence.Thus, P1 serves as a reminder that the infectious disease phenotype requires specific microbial exposures on a permissive immunologic background.
Fig. 2 The p.R249* allele impairs signaling through TYK2.A TYK2 activation in response to No stimulation (NS), IFNα/β, and IL-10 in LCL from HC and P1.Whole-cell lysates were harvested for immunoblotting of total TYK2 and tyrosine-phosphorylated TYK2 (pTYK2).GAPDH was used as the loading control.The bar graph shows the fold change in tyrosine phosphorylation with reference to total protein.The figure is representative of two experiments with mean and standard error of the mean depicted.B STAT phosphorylation in response to IFNα/β, IL-10, or IL-23 stimulation of LCL from HC or P1, as determined by flow cytometry.MFI, mean fluorescence intensity.The cells were stimulated with IFNα/β (105 IU/ml), IL-10 (50 ng/ml), or IL-23 (100 ng/ml) for 30 min.C Phosphorylation of STAT1 and STAT2 (in response to IFNα/β) in A549 WT or in A549 TYK2 −/− transfected with EV (empty vector), TYK2 WT, or TYK2 p.R249*.D Western blot determination of phospho-p38 (pp38) and total p38 in the whole lysate of A549 WT or of A549 TYK2 −/− cells that were transfected with either empty vector (EV), TYK2 WT, or TYK2 p.R249*.Densitometry for pp38 protein levels normalized to total p38 levels.GAPDH used as a loading control and TYK2 as a control of transfection in A549 TYK2 −/ .− .Data represent the means from triplicates, and the results are representative of at least three independent experiments.*p < 0.05 ◂ TYK2's scaffolding function is required for the stable surface expression of IFNAR1, IL10Rb, and IL12Rb1 [3,[35][36][37][38][39].We observed diminished cell surface expression of these proteins, along with decreased intracellular levels, likely due to their instability from loss of TYK2 and their subsequent degradation.On the other hand, IFNAR2 is present in comparable quantities at the cell surface between P1 and controls, consistent with its lack of binding/scaffolding activity by TYK2.Attempts to restore TYK2, with translational read-through of the premature stop codon and chaperone treatment with 4PBA, were unsuccessful and did not improve type I IFN signaling (data not shown).Similarly, treatment with IFN-γ only had modest correction of select ISG.Collectively, these findings would argue that pharmacologic doses of IFN may be futile clinically and that genetic-based therapy or hematopoietic stem cell transplantation may be urgently required to correct the immunodeficiency of complete TYK2 deficiency.

Fig. 1
Fig. 1 Novel homozygous TYK2 variant c.745C > T (p.R249*), leading to TYK2 deficiency.A Chromatogram of Sanger sequencing from a healthy control (HC) and the patient (P1) showing the corresponding mutations in TYK2.B Schematic representation of functional domains of the TYK2 protein.R249 (identified by the arrow) depicts the position of the mutation, located in the FERM, which results a new premature stop codon (p.R249*).C Immunoblotting showing

Fig. 3 p
Fig. 3 p.R249* in TYK2 causes loss of scaffolding function and diminished cell surface expression of select cytokine receptors in LCL.IFN-αR1, IFN-αR2, IL-12Rβ1, IL-10Rβ expression determined by flow cytometry.Analysis on live gate either at the cell surface

Table 1
Laboratory findings (H) higher than the upper limit of the reference range, (L) lower than the lower limit of the reference range ** Post-high dose corticosteroids during acute treatment of encephalitis * Post-IVIG ** Had received a dose of rituximab 3 months prior *