Genomic sequencing and functional analyses identify MAP4K3/GLK germline and somatic variants associated with systemic lupus erythematosus

Objectives MAP4K3 (GLK) overexpression in T cells induces interleukin (IL)-17A production and autoimmune responses. GLK overexpressing T-cell population is correlated with severity of human systemic lupus erythematosus (SLE); however, it is unclear how GLK is upregulated in patients with SLE. Methods We enrolled 181 patients with SLE and 250 individuals without SLE (93 healthy controls and 157 family members of patients with SLE) in two independent cohorts from different hospitals/cities. Genomic DNAs of peripheral blood mononuclear cells were subjected to next-generation sequencing to identify GLK gene variants. The functional consequences of the identified GLK germline or somatic variants were investigated using site-directed mutagenesis and cell transfection, followed by reporter assays, mass spectrometry, immunoblotting, coimmunoprecipitation, and in situ proximity ligation assays. Results We identified 58 patients with SLE from Cohort #1 and #2 with higher frequencies of a somatic variant (chr2:39 477 124 A>G) in GLK 3′-untranslated region (UTR); these patients with SLE showed increased serum anti-double-stranded DNA levels and decreased serum C3/C4 levels. This somatic variant in 3′-UTR enhanced GLK mRNA levels in T cells. In addition, we identified five patients with SLE with GLK (A410T) germline variant in Cohort #1 and #2, as well as two other patients with SLE with GLK (K650R) germline variant in Cohort #1. Another GLK germline variant, A579T, was also detected in one patient with SLE from Cohort #2. Both GLK (A410T) and GLK (K650R) mutants inhibited GLK ubiquitination induced by the novel E3 ligase makorin ring-finger protein 4 (MKRN4), leading to GLK protein stabilisation. Conclusions Multiple GLK germline and somatic variants cause GLK induction by increasing mRNA or protein stability in patients with SLE.


Human samples
This study was conducted in accordance with the Helsinki Declaration. A total of 431 individuals, including 250 healthy/non-SLE individuals and 181 SLE patients were enrolled in this study (Cohort #1 and Cohort #2). No Taiwanese aborigines were enrolled in both cohorts. For Cohort #1, 163 non-SLE individuals (6 healthy controls and 157 non-SLE family members of SLE patients) and 101 SLE patients were referred to the Division of Immunology and Rheumatology at Taichung Veterans General Hospital in Taichung City, Taiwan. Briefly, 77 SLE patients and 157 non-SLE family members from 62 families were randomly enrolled to Cohort #1. Among these 62 families, 15 families each has two SLE patients. To enhance statistical power, 24 sporadic SLE patients and 6 healthy participants were further recruited to Cohort #1. All SLE patients fulfilled the 1997 ACR Revised Criteria for Classification of Systemic Lupus Erythematosus. 1 Disease activity was determined by SLEDAI score. 1 For Cohort #2, 87 healthy individuals and 80 sporadic SLE patients were referred to the Division of Rheumatology at Kaohsiung Medical University Hospital in Kaohsiung City, Taiwan. Unlike Cohort #1, Cohort #2 did not recruit any individuals from the same family. All SLE patients fulfilled the 1982 Revised Criteria for Classification of Systemic Lupus Erythematosus. 2 Disease activity was determined by SLEDAI score. 2 All study participants provided written informed consent. Peripheral blood collections from healthy controls and patients, as well as experiments were approved by the ethical committee of Taichung Veterans General Hospital (#C10130, #SE17193B) and of Kaohsiung Medical University Hospital (KMUHIRB-E(I)-20190108).

Next-generation sequencing (NGS)
Ten ng genomic DNAs from PBMCs were used for multiplex PCR of a panel covering the exon regions of the human map4k3 gene (Ion AmpliSeq Custom Panel, Life Technologies). Library construction of the amplicons and subsequent enrichment of the sequencing beads were performed using Ion Torrent Personal Genome Machine (Life Technologies) according to the manufacturer's protocol by Mission Biotech. DNA sequence data were analyzed by Variant Calling using built-in software Torrent Suite v5.10 and Partek Flow v10.0.21.0509. NGS using Ion Torrent system or Illumina system may have their respective weak points; nevertheless, the data derived from these two systems show a strong correlation (~0.93-0.97). 3

Cell lines and transfection
The human Jurkat T leukemia cell line (ATCC, TIB-152) was cultured in RPMI-1640 medium; human embryonic kidney cell line (HEK293T; ATCC, CRL-11268) was cultured in DMEM medium. Both media were supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 mg/ml streptomycin (Gibco). All cells were free of mycoplasma contamination and grown at 37°C in a humidified atmosphere of 5% CO 2 in air. Plasmids were transfected into HEK293T cells using polyethylenimine reagents (Sigma). Plasmids were transfected into Jurkat T cells by electroporation with the Neon Transfection System (Invitrogen) on 1,430 V for a duration of 30 ms and 1 pulse.

Luciferase reporter assays
Luciferase reporter assays for GLK 3'-UTR activity were performed using the Secrete-Pair™ Dual Luminescence Assay kit (GeneCopoeia) according to the manufacturer's instructions. 5 The 1.352-kb GLK 3'-UTR-driven gaussia-luciferase reporter plasmid containing the secreted alkaline phosphatase (SEAP) internal control was transfected into 5x10 6 Jurkat T cells. After 24 h, 10 μl Tcell supernatants were collected and then incubated with the gaussia luciferase substrate (100 μl GL-H buffer, GeneCopoeia) for 30 s. For internal control, 10 μl T-cell supernatants were incubated at 65 °C for 5 min, then incubated with the alkaline phosphatase (AP) substrate (100 μl AP buffer, GeneCopoeia) for 5 min. The levels of gaussia luciferase (Luc) were normalized to that of AP.

In situ proximity ligation assay (PLA) technology
In situ PLA assays were performed using the Duolink In Situ-Red kit (Sigma-Aldrich) according to the manufacturer's instructions as described previously. 4,6 Briefly, Flag-GLK plus Myc-MKRN4-cotransfected HEK293T cells were fixed and permeabilized. Samples were incubated with anti-Flag (#ARG66332, Arigo biolaboratories) and anti-Myc (clone 9E10) monoclonal antibodies, followed by species-specific secondary antibodies conjugated with oligonucleotides (PLA probes, Sigma-Aldrich). After ligation and amplification reactions, PLA signals from each pair of PLA probes in close proximity 7 (< 40 nm; MKRN4-GLK interaction) were visualized as individual red dots and analyzed by Leica DM2500 upright fluorescence microscope. Red dots represent direct interaction signals.

Immunoprecipitation and immunoblotting analysis
Immunoprecipitation was performed by incubation of 1.0 mg protein lysates with 20 μl anti-Flag or anti-Myc Sepharose beads (Merck) at 4 °C for 3 h. The immunocomplexes were washed with lysis buffer (50 mM Tris-HCl, 0.2% NP40, 125 mM NaCl, 1.5 mM MgCl 2 , 5% glycerol, 25 mM NaF, and 1 mM Na 3 VO 4 ) three times at 4 °C, followed by boiling in 5x loading buffer at 95 °C for 3 min. The immunoblotting analyses were performed as described previously. 4,6 Liquid chromatography-mass spectrometry and data analysis For identification of GLK-interacting proteins, immunocomplexes of Flag-tagged GLK were immunoprecipitated by anti-Flag antibody (clone M2; Merck) from lysates of Jurkat T cells transfected with vector or Flag-GLK plasmid. For identification of MKRN4-induced GLK ubiquitination residues, immunocomplexes of Myc-tagged MKRN4 were immunoprecipitated by anti-Myc antibody (9E10; Sigma) from lysates of Jurkat T cells transfected with Myc-MKRN4 plasmid. Protein bands were excised from Instant blue (GeneMark)-stained SDS-PAGE gels. Proteins were digested with trypsin and subjected to LC-MS/MS analyses by LTQ-Orbitrap Elite hybrid mass spectrometer using approaches described previously. 4,6 The peptide data were analyzed by MASCOT MS/MS Ions Search (Matrix Science) under the following condition: peptide mass tolerance, 20 ppm; fragment MS/MS tolerance, 2 Da; allow up to 1 missed cleavage; peptide charge, 2 + , 3 + , and 4 + .

Statistical analyses
The normality of each column data was determined by Kolmogorov-Smirnov and Shapiro-Wilk tests using SPSS 25 software. The association of somatic variants with SLE or individual symptoms/treatments was determined by Chi-square test and Fisher's exact test. The statistical significances between two unpaired groups were analyzed using Student's t-test for normally distributed data. All statistical analyses of clinical data were further independently verified by 2 biostatisticians.

Patient and public involvement
Patients or the public were not involved in the design, or conduct, or reporting, or dissemination plans of our research.    S9 S12 S15 S6 S11 S3 S13 S14 BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s)