Hypomorphic biallelic LIFR variants define a multisystem disorder with survival beyond infancy and hyperinflammatory vulnerability.

Hypomorphic biallelic LIFR variants define a multisystem disorder with survival beyond infancy and hyperinflammatory vulnerability

Abstract

Background

The leukemia inhibitory factor receptor (LIFR) is a key signaling receptor within the IL-6 cytokine-family, and its integrity is essential for STAT3 and ERK activation in developmental and immune pathways. Recessive, predominantly truncating variants in LIFR cause Stüve-Wiedemann syndrome (SWS), historically a neonatal-lethal skeletal dysplasia. As more individuals survive beyond infancy, the clinical spectrum and its biological basis remain incompletely defined.

Methods

We describe the clinical heterogeneity with considerable survival beyond infancy and phenotypic expansions in neurodevelopment and immune dysregulation and determine how missense variants alter cytokine and receptor trafficking. To define the expanded disease spectrum and underlying mechanisms, we assembled an international cohort of 22 previously unreported individuals with biallelic LIFR variants, combined with functional characterization of missense alleles using cellular receptor abundance and STAT/ERK signaling assays, and proteomic profiling of patient-derived bronchoalveolar lavage (BAL) cells.

Results

We identified a markedly expanded phenotype among survivors beyond infancy, including neurodevelopmental disorders, allergy, and hyperinflammatory courses, including COVID-19–associated multisystem inflammatory syndrome in children (MIS-C). In cellular assays, the hypomorphic missense variant p.Cys270Gly showed reduced cell-surface LIF-receptor abundance, STAT/ERK phosphorylation and cellular proliferation upon LIF stimulation. Analysis of additional missense variants revealed functional domain-specific effects: N-terminal extracellular variants retained partial activity, whereas C-terminal cytoplasmic variants showed near-complete loss-of-function. Patient bronchoalveolar-cell proteomics identified dysregulations in endolysosomal recycling of defective LIF-receptor.

Discussion

Our cohort’s findings may position LIFR deficiency as a survivable multisystem disorder with relevant neurological and autoimmune-inflammatory vulnerability. Variant-level functional stratification and proteomic signatures provide a coherent biological framework to support risk assessment and long-term clinical management in affected individuals.

Keywords: ERK signaling, genotype-phenotype correlation, inborn errors of immunity, intracellular trafficking, JAK/STAT 3 signaling, LIFR deficiency, MIS-C, neurodevelopmental disorders

Introduction

Stüve-Wiedemann syndrome (SWS) was first described in two patients (1) with characteristic features of bone abnormalities, short stature, episodic hyperthermia, respiratory distress, and feeding difficulties. Biallelic variants in the leukemia inhibiting factor receptor gene (LIFR) were identified as a cause for Stüve-Wiedemann syndrome type 1 (2) More recently, biallelic variants in the interleukin-6 cytokine family signal transducer gene (IL6ST/gp130) were associated with a similar phenotype named Stüve-Wiedemann syndrome type 2 (3). Loss-of-function variants in any of these genes lead to a perturbation in interleukin-6 signal transduction that may affect intracellular Janus kinase (JAK) phosphorylation as well as activation and nuclear translocation of signal transducers and activators of transcription protein (STAT) (4). Previous basic science research indicated the role of LIFR in the function and development of post-mitotic plasmacytoid dendritic cells (5) as well as matured cortical neurons (6), both found in post-mitotic tissue. Recent evidence identified endosomal internalization and endolysosomal trafficking of LIFR for subsequent degradation (7, 8).

Genotype-phenotype correlations for pathogenic IL6ST variants have shown location-dependent perturbations in domain function and, ultimately, defective cytokine response (4, 9, 10). While patients with LIFR-related SWS have predominantly been reported with infantile death, there is a lack of detailed phenotypic characterization of the multi-system involvement in this syndromic immunodeficiency, particularly in “long-term survivors” after infancy (11–13).

In this study, we set out to i) report 22 novel patients with a spectrum of LIFR variants and variable phenotypic progression, ii) examine the LIFR genotype-phenotype spectrum with hypomorphic patient alleles in cell assays to examine domain-specific loss-of-function, and iii) investigate dysregulated endolyosomal trafficking downstream of defective LIF-receptor which is particularly relevant for the phenotype in postmitotic tissues including skeleton, neurons and immune cells.

Materials and methods

Patient characterization and genotype-phenotype correlation analyses

The patients were recruited by multi-center study sites via GeneMatcher, ERN ITHACA or ESNEK, each with regional IRB approval (20-1711, Medical Faculty of University Cologne, Germany) (14). To supplement our analyses on allelism, we conducted a genotype-phenotype correlation study in 74 patients with variants in LIFR of which n=22 are novel patients collected in our cohort and identified domain-specific phenotype clustering (Supplementary Material) (2, 9, 15–37).

Patient recruitment

We examined publications in PubMed for patients with LIFR-related SWS from 01.01.2010 to 01.09.2023 and identified 24 publications with 52 patients (2, 9, 15–33, 35–37).

We re-evaluated cases of cohorts with n>5 cases regarding the core features immunodeficiency, skeletal dysplasia, and neurological disorders (neurodevelopmental disorders, muscular hypotonia, neurodegenerative courses). For all indicated percentages below, we identified the reported phenotypes from published patients and novel patients from our cohort. Unreported phenotypes in patients were designated as ‘not defined’ (ND). The reported percentages below present the percentage of manifested phenotypes from the total number of patients.

Cloning

The hLIFR (P42702) in pcDNA3.1 vector was used as a template for side directed mutagenesis using the primer: C270G fw 5’ CAT AAC ATT TGG TTG TGT GAG TCA AG, C270G rev 5’ TCT GAG CCT ACA AGT ATC AC. Gene coding for wildtype and LIFR variants p.Asn128Lys, p.Pro210Cys, p.Cys270Gly, p.Thr646Asn, p.Asn1096Lys, and p.Tyr697Ter were then inserted into pMOWS retroviral vector including hygromycin B resistance.

Cells, reagents and recombinant proteins

The generation of Ba/F3-gp130 cells was described elsewhere (38). The packaging cell line Phoenix-Eco was received from Ursula Klingmüller (DKFZ, Heidelberg, Germany). HEK293T (ACC-635) cells were purchased from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Culture (Braunschweig, Germany). All cells were grown at 37 °C with 5% CO2 in a water-saturated atmosphere in Dulbecco`s modified Eagle`s medium (DMEM) high-glucose culture medium (GIBCO®, Life Technologies, Darmstadt, Germany) with 10% fetal calf serum (GIBCO®, Life Technologies) and 60 mg/l penicillin and 100 mg/l streptomycin (Genaxxon Bioscience GmbH, Ulm, Germany). Murine Ba/F3-gp130 cells were obtained from Immunex (Seattle, WA, USA) and grown in the presence of the fusion protein Hyper-IL-6 (HIL-6). 0.2% (10ng/ml) conditioned medium from a stable clone of CHO-K1 cells secreting HIL-6 in the supernatant (39). HIL-11-Ts was expressed and purified as reported elsewhere (40). Recombinant human LIF (catalog no. 7734-LF) was purchased from R&D Systems (Minneapolis, MN, USA).

Western blotting

50 µg total protein were loaded per lane and separated by SDS-PAGE under reducing conditions and transferred to a nitrocellulose membrane (Amersham Protan; Cytiva; LC, United Kingdom; catalog no. 10600016). Blocking of membrane was performed with blocking buffer (Intercept® Blocking Buffer; LI-COR; USA; catalog no. 927-60001) diluted 1:3 in TBS (10 mM Tris-HCl pH 7.6, 150 mM NaCl) for 1 h. Primary antibodies (STAT1; catalog no. 9172; phosphor-STAT1; catalog no. 9167; Phospho-STAT3; Tyr-705; D3A7; catalog no. 9145; STAT3; 124H6; catalog no. 9139; ERK1/2; catalog no. 9102; pospho-ERK1/2; catalog no. 4370, Cell Signaling Technology, USA) and (LIFR; catalog no. BAF249, R&D Systems, USA) were diluted 1:1,000 in blocking buffer containing 0.2% Tween-20 (Sigma-Aldrich; USA; catalog no. P1379-1L) for at least 90 min at ambient temperature or overnight at 4 °C. Membranes were washed with TBS-T (0.1% Tween-20) and then incubated with secondary fluorophore-conjugated antibodies 1:10,000 (IRDye® 800CW Donkey anti-Rabbit; catalog no. 926-32213, IRDye® 680RD Donkey anti-Mouse; catalog no. 926–68072 and Donkey anti-Goat; catalog no. 926-32214, LI-COR; USA) for 1 h. Signal detection was achieved using LI-COR Odyssey; USA; Model 2800). Secondary antibodies were detected simultaneously on different channels. Data analysis was conducted using Image Studio Lite 5.2.

Cell viability assay

Ba/F3-gp130 cells were washed three times with PBS to remove cytokines from the medium. Cells were suspended in DMEM containing 10% fetal calf serum, 60 mg/l penicillin and 100 mg/ml streptomycin at a density of 5x104 cells/ml. Cells were cultured for three days in a volume of 100 µl with or without cytokines or inhibitor in the indicated concentrations. The CellTiter Blue Viability Assay (Promega, Karlsruhe, Germany) was used to determine the approximate number of viable cells by measuring the fluorescence (Λex560 nm/Λem590 nm) using the Infinite M200 Pro plate reader (Tecan, Crailsheim, Germany). After adding 20 µl/well of CellTiter Blue reagent (time point 0), fluorescence was measured after 60 minutes every 20 min for up to two hours. For each condition of an experiment, three wells were measured. All values were normalized by subtracting time point 0 values from the final measurement.

Transfection and transduction of cells

Ba/F3-gp130 cell lines were retrovirally transduced with the pMOWS expression plasmids as described previously (41). Transduced cells were grown in DMEM medium as described above supplemented with 10 ng/ml HIL-6. Selection of transduced Ba/F3-gp130 cells was performed with hygromycin B (1 mg/ml) (Carl Roth, Karlsruhe, Germany) for at least 2 weeks. Afterwards, the generated Ba/F3-gp130 cell lines were analyzed for receptor cell surface expression via flow cytometry. HEK293T cells were transfected by 7.5 µg pDNA with 15 µl TurboFect (Thermo Fisher, USA, catalog no. R0532) and incubated for 48 h before cell lysis and Western Blotting.

Stimulation assay

Ba/F3-gp130 cell lines were washed three times with PBS to remove cytokines and starved in serum-free DMEM for 3h. Cells were stimulated for 15 min with LIF (10 ng/ml) or HIL-11-Ts (50 ng/ml), harvested, frozen in liquid nitrogen and then lysed. Cells were lysed for 45 min with buffer containing 10mM Tris-HCl, pH 7.5, 150mM NaCl, 0.5mM MgCl2, 1mM Na3VO4 and a complete EDTA-free protease inhibitor mixture tablet (Roche Diagnostics, Mannheim, Germany). Protein concentration was determined by a BCA protein assay (Thermo Fisher Scientific) according to the manufacturer’s instruction. Receptor expression or pathway activation was then analyzed by Western Blotting which is described in detail in the Supplementary Material.

Cell surface detection of cytokine receptors via flow cytometry

Cell surface expression of stably transfected Ba/F3-gp130 cell lines was detected by specific antibodies. 5×105 cells were washed in FACS buffer (PBS, 1% BSA) and then incubated in 50 µl of FACS buffer containing the indicated specific primary antibody (anti-LIFR; 1:20 diluted; catalog no. BAF249, R&D Systems; USA). After incubation of 1 h at room temperature, cells were washed and resuspended in 50 µl of FACS buffer containing secondary antibody (NothernLights 493–conjugated anti-goat IgG 1:200; catalog no. NL003, R&D Systems; USA) and incubated for 30 min at room temperature. Cells were washed and resuspended in 500 µl of FACS buffer and analyzed by flow cytometry (BD FACSCanto II flow cytometer using the FACSDiva software, BD Biosciences). Data analysis was conducted using FlowJo Version 10 (Tree Star Inc, US).

Please see Supplementary Material for detailed methods on cloning, cells, reagents and recominant proteins, cell viability assay, and transfection and transduction of cells.

Bronchoalveolar cell sampling and LC-MS/MS proteomics

We conducted LC-MS/MS proteomics on bronchoalveolar cells from a patient with LIFR:p.Cys270Gly (patient 1) and a healthy sex- and age-matched control subject, both samples obtained by flexible bronchoscopy. The bronchoscope was meticulously wedged in the lingula, and a total of four 20 ml aliquots of normal saline, prewarmed to 37 °C, were sequentially instilled and manually aspirated into the same syringe until no further fluid was retrieved. Total recovery rates approximated 70% in both patients. The first aliquot was used for microbiological analysis while the remaining portions were pooled for subsequent investigations. The lavage fluid was promptly transported to the laboratory at room temperature where it was centrifuged down with 400 rpm over 10 min at 4 °C for decanting of supernatant over three times. The cells were then frozen in -70 °C until further use.

We washed bronchoalveolar cells with PBS for five times. Lysis buffer (6 M guanidinium chloride, 2.5 mM TCEP (tris(2-carboxyethyl) phosphine), 10 mM CAA (chloroacetamide), 100 mM Tris-HCl) was added to each sample. Samples were heated at 95 °C for 5–10 min, lysed with the Bioruptor (30-s sonication, 30-s breaks, 10 cycles, 2–3 repeats) and centrifuged at 20,000g for 20 min. Then, 2 µL of the protein supernatant was diluted at least 10 times with 20 mM Tris to 20 µl to reduce the concentration of GuCl to lower than 0.6 M. Protein concentration was then measured by Nano Drop. Samples were then diluted 10 times and digested with trypsin (1:50, Promega, Mass Spec Grade) overnight at 37 °C. The digestion was stopped by adding 50% of FA to 1% (final concentration). Peptides were separated on a 25-cm, 75-μm internal diameter PicoFrit analytical column (New Objective) packed with 1.9 μm ReproSil-Pur 120 C18-AQ medium (Dr. Maisch) using an EASY-nLC 1000 (Thermo Fisher Scientific). The column was maintained at 50 °C. Buffer A and B were 0.1% formic acid in water and 0.1% formic acid in 80% acetonitrile. Peptides were separated on a segmented gradient from 6% to 31% buffer B for 120 min and from 31% to 50% buffer B for 10 min at 200 nL min–1. Eluting peptides were analyzed on a QExactive HF mass spectrometer (Thermo Fisher Scientific). Peptide precursor m/z measurements were carried out at 60,000 resolution in the 300 to 1,800 m/z range. The top 10 most intense precursors with charge state from 2 to 7 only were selected for HCD fragmentation using 25% normalized collision energy. The m/z values of the peptide fragments were measured at a resolution of 30,000 using a minimum AGC target of 8 × 103 and 55-ms maximum injection time. Upon fragmentation, precursors were put on a dynamic exclusion list for 45 s. The raw data were analyzed with MaxQuant version 1.5.2.8 using the integrated Andromeda search engine (42). Peptide fragmentation spectra were searched against the canonical and isoform sequences of the H. sapiens and M. musculus reference proteomes. The digestion parameters were set to ‘specific’ and ‘Trypsin/P,’ The minimum number of peptides and razor peptides for protein identification was 1; the minimum number of unique peptides was 0. Protein identification was performed at a peptide spectrum matches and protein false-discovery rate of 0.01. The ‘second peptide’ option was on. Successful identifications were transferred between the different raw files using the ‘Match between runs’ option. Label-free quantification (LFQ) was performed using an LFQ minimum ratio count of 2. LFQ intensities were filtered for at least four valid values in at least one group and imputed from a normal distribution with a width of 0.3 and down shift of 1.8. Differential expression analysis was performed using Limma’s moderated two-sided t-test. The adjusted P values, using Benjamini–Hochberg’s procedure, were used for further analysis and visualization. Gene Ontology category enrichment analysis of the significantly dysregulated proteomic datasets was done using Shiny GO server as demonstrated in the Figure legends (43).

Statistical analysis

Data are provided as arithmetic means ± SEM using GraphPad Prism, Version 8. Statistically significant differences between two groups were determined with a Students’ t-test, including Welch’s correction if indicated. Statistically analysis between several groups were determined using a two-way ANOVA, including Tukey correction. Significance was calculated as follows p > 0.05: n.s.; p < 0.05: *; p < 0.01: **; p < 0.001: ***; p < 0.0001: ****. In vitro assays were performed at least in three independent experiments.

Results

Phenotype expansion and survival beyond infancy with hypomorphic alleles

General features

Here, we report 22 novel patients from 20 families (Table 1). The female-to-male ratio was at 1.4 (females 13, males 9). The last reported age of the patients ranged from fetal demise at 28th week of gestation until 20 years. While 15 patients were reported as alive on last examination, seven patients were reported with an early demise, with the age at death ranging from in utero (28th week of gestation) to 7 years. Consanguinity was found in 73% (16/22). The ethnicities of the patients included Arabic (16/22), Afghan (1/22), European (2/22), Turkish (1/22), Indian (1/22), and Asian Indian (1/22).

Table 1Patient IDs are indicated as P# for the newly described cohort, with the corresponding #-number referring to the same individual in the complete cohort of 74 patients for genotype-phenotype correlation study listed in Supplementary Table 1 (P1-P22 and as #50–67 and #71–#74).

Patient P5, #54 P19, #71 P6, #55 P7, #56 P8, #57 P9, #58 P10, #59 P11, #60 P12, #61 P13, #62 P14, #63 P15, #64 P17, #66 P18, #67 P20, #72 P21,#73 P1, #50 P16, #65 P4, #53 P3, #52 P2, #51 P22, #74

Domain D1 CBM1 D2 CBM1 D3 Ig-like D4 CBM2 D5 CBM2 D8 (FNIII) NA

cDNA c.194G>C c.653dup c.756dup c.756dup, c.2444C>G c.808T>G c.1122-1_1122insAAAG c.1438-4A>G c.1469C>G c.2497 + 1G>A Deletion of LIFR exons 17-20

Protein p.Cys65Ser p.Glu219GlyfsTer3 p.Lys253Ter p.Lys253Ter,p.Thr815Arg p.Cys270Gly p.Ser374ArgfsTer9p p.? p.Ser490Ter p.? NA

Zygosity Homozygous Homozygous Homozygous Compound heterozygous Homozygous Homozygous Homozygous Homozygous Homozygous Homozygous

gnomAD N N N N N N N N N N N N N N N N; 1/152134 N N 1/151968 N N NA

Consanguinity Y Y Y Y Y Y Y Y N N Y Y N Y Y N Y Y Y N N Y

Sex F F F M F M M M M F F F F F F M F M F M M F

Ethnicity Arabic Arabic Arabic Arabic Arabic Arabic Arabic Arabic Arabic Arabic Arabic Arabic Arabic Arabic Romanian Romanian Afghan Arabic Turkish Indian Asian Indian Dari/Persia

Age of last follow-up 9m 7y 8y 7y 3m 4y 4y 2m 20m 20m 20y 18y 3y 6m 3m 4y 8m 7y 6y 8m 79d 15y In utero 28 gest. w 19d 5y 10m

Alive or dead Alive Alive Alive Died 7y Died 3m Alive Alive Died 2m Alive Alive Alive Alive Alive Alive Died 4y 8m Alive Died 6y8m Died 79d Alive Fetal death Alive Alive

Family N SWS SWS SWS SWS N SWS SWS SWS SWS SWS SWS SWS SWS SWS SWS SWS SWS SWS SWS SWS SWS

Perinatal features

Oligohydramnios N N ND N ND N N Y N N ND ND Y N Y ND N N N N N ND

Ophthalmological N Y Y Y ND Y Y ND Y Y Y Y Y N Y N Y N ND N N ND

Craniofacial Y Y Y Y ND Y Y ND Y Y Y Y Y N Y N Y Y Y Y Y Y

Muscular hypotonia Y Y ND N ND Y ND Y Y Y ND ND N N Y Y Y Y Y NA Y Y

Hyporeflexia N Y Y Y ND Y Y ND ND ND Y Y Y ND Y Y N N N NA ND N

Disease courses

NDD Motor Motor, speech Motor, speech Motor, speech, cognitive N/A Motor, cognitive Motor, speech, cognitive N/A Motor, speech Motor, speech Speech Speech N ND Motor, speech, cognitive Motor, speech Motor, speech, cognitive Motor Motor, speech NA ND Motor, speech, cognitive

Neurodegeneration N N N Y N Y N N N N N N N N Y N Y N N NA ND N

Dysautonomia N Y N Y N Y N N Y Y N N Y Y Y N Y Y Y NA ND Y

Feeding difficulties Y Y Y Y N Y Y Y Y Y Y Y Y Y Y Y Y Y Y NA Y Y

Immunodeficiency Y Y N Y Y Y N N N Y Y Y Y Y Y Y Y Y N NA Y Y

Allergies N N N Y Y N Y N Y Y Y Y ND ND Y N N N N NA N N

Skin N Y N N N N N N N N Y Y Y ND Y Y N N N ND N Y

Pulmonary Y Y N Y Y Y N Y Y Y Y Y Y Y Y N Y Y N ND Y Y

Cardiac Y N Y Y N N Y N N Y Y N ND ND N N Y Y N ND Y N

Short stature Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y N Y ND ND Y

Skeletal Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y N N Y Y Y

Kidney N Y N Y N Y N ND ND N Y ND ND ND N N Y N N ND ND N

Gastrointestinal Y Y N N N N N N N N N N ND ND N N N ND N Y N Y

Endocrinological N Y ND N ND N ND ND N N N N ND ND N Y N ND N ND N N

Cohort of 22 novel patients with LIFR-related disorders with variants on gene and protein, consanguinity, sex, ethnicity, age at last follow-up examination, status alive or dead, family history of SWS, oligohydramnios, ophthalmological features, craniofacial dysmorphia, muscular hypotonia, hyporeflexia, neurodevelopmental disorders (NDD), neurodegenerative features (spasticity, epilepsy), dysautonomia, feeding difficulties, immunodeficiency, allergies, skin features, pulmonary features, cardiac features, short stature, skeletal features, kidney features, gastrointestinal features, and endocrinological features. 1. Homozygous LIFR variants according to NM_001127671.2, ENST00000453190.7. Protein LIFR mutations according to NP_001121143.1, ENSP00000398368.2. In P3, the parents were first cousins, each heterozygous for c.1469C>G; p.Ser490Ter, fetal testing after in utero death was not performed according to parental wishes, (and the molecular genetic reported this LIFR variant as pathogenic as previously reported cases had been annotated as pathogenic in the ClinVar database (variation ID: 1070966)). Population database counts according to gnomAD v3.1.2. M, male; F, female, y, years; m, months; d, days; Y, yes; N, no; ND, not defined; NA, not available (due to fetal death).

Immunological disorders

Primary immunodeficiency was identified in 73% of cases (16/22) and presented with recurrent bacterial, fungal and viral lower respiratory tract infections (12/22, 55%), eye (7/22, 32%) and skin infections (5/22, 23%). Dental decay was noted in three patients. Atopic diseases were present in 36% of cases (8/22), mostly presenting with allergic asthma. Skin abnormalities were found in 27% of cases (6/22) and included xerosis cutis, seborrheic dermatitis, and delayed wound healing.

Patient P1 carrying a homozygous variant in p.Cys270Gly had a COVID-19 infection that led to acute cardiorespiratory failure within 12 hours of symptom onset, failed resuscitation in the emergency room, and death at six years and eight months of age. Patient P13 with a homozygous variant in p.Glu219GlyfsTer3 presented with COVID19-related multisystem inflammatory syndrome in children (MIS–C) at the age of 20 months that led to acute cardiorespiratory failure with resuscitation, acute kidney injury, and coagulopathy.

Skeletal abnormalities

Four cases died in utero or during infancy. Skeletal features were observed in all cases (22/22) and included bowing of the long limbs in 16 cases, camptodactyly in 13 cases, scoliosis in eight cases, osteopenia in four cases, hip dysplasia in four cases, shortened upper and/or lower limbs in four cases (Figures 1D, H, I–O). Short stature was found in 86% of cases (19/22). Moreover, those deformities often progressed throughout life and resulted in several orthopedic surgeries and/or immobility.

Neurological disorders

Neurodevelopmental disorders (NDD) were found in 73% of cases (16/22), most frequently with motor delay (15/22, 68%), speech delay (13/22, 59%), and intellectual disability (6/22, 27%). Of note, two patients only had speech delay but age-adequate motor and cognitive milestones. In two patients carrying the LIFR variants p.Cys270Gly and p. Lys253Ter, we observed corpus callosum dysgenesis, brain atrophy and multiple FLAIR-hyperintensities on brain magnetic resonance imaging (MRI) (Figures 1F, G). Neurodegenerative features were present (4/22, 18%) and included spastic paraplegia and epileptic encephalopathy. Dysautonomia was present in 55% of cases (12/22) and included tongue ulcerations (n=2), hyperthermia (n=6), hypothermia (n=1), and paradoxical sweating (n=3). One patient suffered from rhabdomyolysis during a hyperthermic event.

Population genetics and molecular genetic spectrum in milder disease

The genotypes in our patient cohort (n=22) included four truncating variants, two splice site variants, and three missense variants (Table 1; Figure 1A). Only two of the above variants were present in the population database gnomAD v 3.1.2 (c.1438-4A>G with 1/151,968 in heterozygous state and c.2444C>G,p.Thr815Arg with 1/152,134 in heterozygous state; no homozygotes were recorded for any of these variants). The other variants were absent from population databases.

In first analyses, we examined a potential association of the predicted variant effect on protein (missense vs. truncation) with patient survival. Seven patients were reported as deceased. We observed homozygous truncating variants in six of these patients and a homozygous missense variant in one patient.

Upon analysis of the variant localization within specific protein domains, we observed that patients with variants affecting the C-terminal half of the extracellular region showed early demise, with age of death ranging from 28th week of gestation (p.Ser490Ter), to 19 days (c.2497 + 1G>A) and 79 days postnatally (p.Ser374ArgfsTer9), respectively. The patient with in utero death and fetal akinesia is shown in Figure 1B (left) and 1C. A 15-year-old patient with a variant in D5 CBM2 (c.1438-4A>G) had severe life-threatening events of paradoxical sweating requiring intensive care treatment. The patient with a variant in D4 CBM2 (p.Ser374ArgfsTer9) presented with an episode of severe rhabdomyolysis upon infection at 79 days of age. The Supplementary Material shows a genotype-phenotype correlation analysis with all published patients.

N-terminal LIFR missense variants reduce and C-terminal variants abolish protein function

As we observed several cases where early death was associated with variants in the C-terminal half of the extracellular region, we examined whether the localization of missense variants had any effect on functional activity.

In Ba/F3-gp130 cellular proliferation assays, stimulation with LIF (0.00001–100 ng/ml) showed reduced proliferation for all LIFR mutants up to 0.01 ng/ml except for p.Arg88His when compared to Ba/F3 cells expressing the wild-type proteins (Figure 2A). With LIF stimulation above 0.01ng/ml, all missense variants in the extracellular regions showed markedly reduced proliferation, whereas variants in the cytoplasmic region did not proliferate at all. EC50 levels were elevated for LIFR p.Asn128Lys and LIFR p.Gly779Val (Figure 2B).

In Ba/F3-gp130 cells expressing LIFR variants with mutations in the extracellular region, maximal signal transduction was largely unaffected (Figure 2C). However, basal activity was significantly reduced with LIFR variants in the extracellular region p.Asn128Lys, p.Pro210Cys, p.Thr646Asn, and p.Gly779Val, but notably unremarkable with p.Arg88His in comparison to wild type (Figure 2D). This finding suggests that missense variants in the N-terminal part of the extracellular region may not have a severe effect on basal activity which correlates with the less severe phenotype in patients with N-terminal LIFR variants.

Finally, we examined one missense variant in the cytoplasmic region (p.Asn1096Lys) as well as a deletion of the cytoplasmic region (p.Tyr697Ter). These variants in the cytoplasmic region have notably shown both absent maximum signal transduction and basal activity, causing complete loss-of-function in LIFR. These findings correlate with severe phenotypes and early death in LIFR patients with variants in the C-terminal half of LIFR as well as the noted absence of biallelic variants in the cytoplasmic region which may not be viable in human (Figures 2A–D).

LIFR:p.Cys270Gly missense variant leads to LOF with defective STAT and ERK signaling

Most research on human LIFR deficiency was previously focused on biallelic truncating variants, we examined the biallelic missense mutation p.Cys270Gly in a murine cell model. We picked this particular p.Cys270Gly variant in the D3 Ig-like domain as it was identified in a patient (P1) who survived infancy, gave bronchoalveolar cells during early childhood and later died at 6 years and 8 months of age during a COVID-19 infection. We were interested in this variant as it localized to a protein region which, if mutated, appeared to be associated with better overall life expectancy compared to variants in the D4-D6 domains but associated with a higher risk of infection-associated deterioration and death.

Proliferation of the murine pre-B cell line Ba/F3 is IL-3 dependent (41) The variant p.Cys270 is located in D3 of the LIFR, forming a disulfide bond to p.Cys163 in D2. To investigate the impact of the mutation on cell proliferation we performed a dose-dependent proliferation assay. Ba/F3 cells expressing the wildtype LIFR:gp130 receptor complex proliferate LIF-dose-dependently with a EC50 of 51 pg/ml, whereas Ba/F3 cells expressing the p.Cys270Gly variant proliferate with an EC50 of 639 pg/ml (Figures 3A, B). To analyze the STAT3 phosphorylation, Ba/F3 cells expressing gp130, gp130-LIFR or gp130-LIFR p.Cys270Gly were stimulated with HIL-11 or increasing amounts of LIF. Western blotting showed that the same amounts of LIF (10 ng/ml) led to increased STAT3 phosphorylation, if the wildtype LIFR receptor was expressed, compared to the p.Cys270Gly protein (Figures 3C, D).

With respect to the STAT3 phosphorylation in Ba/F3-gp130 cells expressing the LIFR variants, phosphorylation of STAT1 and STAT3, as well as downstream MAPK/ERK pathway activation were investigated after stimulation with either HIL-11 or LIF. Ba/F3-gp130 cells expressing wildtype LIFR induce STAT3 phosphorylation more efficiently than cells expressing the LIFR p.Cys270Gly variant. Concomitantly with STAT3 phosphorylation also ERK phosphorylation is less efficiently transduced by LIFR p.Cys270Gly. Interestingly, intracellular STAT1 phosphorylation could not be induced by the p.Cys270Gly variant of LIFR, using a high concentration of LIF (Figure 3C).

To investigate total expression, stably transduced Ba/F3-gp130-LIFR and Ba/F3-gp130-LIFR p.Cys270Gly cells were lysed and LIFR was detected by Western blotting, showing the same amount of LIFR p.Cys270Gly compared to the wildtype receptor (Figure 3E). To analyze the density of expressed LIFR on the cell surface, flow cytometry was performed, showing a lower surface presentation of the LIFR p.Cys270Gly variant compared to the wild type (Figure 3F). The hypomorphic p.Cys270Gly variant allows only weak STAT3 phosphorylation but shows a lack of STAT1 phosphorylation upon LIF stimulation.

Patient p.Cys270Gly bronchoalveolar cells show endolysosomal trafficking dysregulation

To explore impact of a LIFR missense variant in patient-derived cells, we conducted LC-MS/MS proteomics on primary patient cells obtained through bronchoalveolar lavage fluid from the patient with the p.Cys270Gly variant and a healthy age- and sex-matched control without Stüve-Wiedemann syndrome (Figure 4A); the patient and control did not have any infection at the point the sample was taken. Of note, TMEM173/STING was upregulated in patient bronchoalveolar cells, already suggesting STING upregulation at a time without clinical infection likely due to chronic hyperinflammation.

We also observed dysregulation of downstream endocytosis trafficking, including significant upregulations in the AP2 subunits AP2A1 and AP2A2, the endosome-associated adaptor protein APPL1 as a Rab5 effector protein, and the Rab5 proteins RAB5A and RAB5C that are involved in maturation of early endosomes to lysosomes. Additional findings in upregulation of endosome-associated Rab proteins include the proteins associated with trafficking from ER to Golgi (RAB1A, RAB1B, RAB6A), trafficking from Golgi to early endosomes (RAB31, AP3D1, GABARAP), trafficking of lipid droplets (RAB18), and trafficking from plasma membrane to endosomes (RAB10, RAB13). A notable finding regarding a stall in late endosomes/multivesicular bodies is indicated by upregulation of CD9 and downregulation of CD63.

Other upregulated Rab proteins indicated a stall in later stages of endolysosomal trafficking, including RAB35 involved in selective autophagic clearance of bacteria (xenophagy) and mitochondria (mitophagy) as well as RAB7A. Notably, VPS11 and LAMP1 were upregulated indicating a stall in autophagolysosomal fusion, while cathepsin L (CTSL), a direct transcriptional target of STAT3, was significantly downregulated, also indicating a stall in autophagolysosomal flux.

We also noted downregulation in immunological proteins including IFNGR1, and NCOA5, as well as in proteins involved in growth including IGFBP3, IGFBP1, and IGF2 (Figure 4B).

Gene Ontology molecular function terms indicated downregulation in the pathways for amino acid responses and the toll-like receptor pathway, and upregulation in pathways for catabolic processes for keratan sulfate, glycosaminoglycans, and aminoglycans (Figures 4C, D).

Discussion

We present the largest cohort of patients with LIFR-related Stüve-Wiedemann syndrome reported to date (n=22), prompting several milder presentations with survival beyond infancy and LIFR missense variants. We leveraged a murine cellular model to assess effects of missense variants in LIFR on cellular proliferation upon stimulation with LIF. While some of the variants were found in patients with mild or moderate phenotypes (p.Thr646Asn, p.Gly779Val, p.Asn1096Lys), we also examined missense variants either in amino acid residues that were previously published with truncating variants, including p.Asn128Lys for p.Asn128LysfsTer2, or located next to published variants, including p.Pro210Cys for the founder variant p.Glu219GlyfsTer3. While the N-terminal variant p.Arg88His did not reveal a significant change in comparison to wildtype, all other variants did indeed confer deleterious effects on cellular proliferation and basal receptor activity of LIFR. To investigate the effects on the cytoplasmic region, we examined a LIFR variant with a large truncation of the cytoplasmic region (p.Tyr697Ter) which displayed an absence of proliferation and basal receptor activity of LIFR. LIF signaling requires a functional receptor complex consisting of LIFR and IL6ST (gp130), with both receptor subunits contributing to downstream signal transduction (44). The absence of a proliferative response observed for the LIFR variant affecting the intracellular domain suggests that the presence of IL6ST is not sufficient to fully compensate for impaired intracellular LIFR signaling. This may indicate that the intracellular domains of both receptor proteins contribute to effective signal transduction, although the precise interplay and relative contributions of LIFR and IL6ST remain to be elucidated (45). When including published reports with LIFR-associated SWS (74 patients from 64 families, Supplementary Material), we observed that cytoplasmic region variants in LIFR were only found in heterozygosity, likely due to its essentiality for cellular viability upon infection (46), and further research may be required to determine whether any homozygous variant in that region may be viable in human.

In addition, our data suggest a tentative association between genotype and phenotype with regards to survival. The seven deceased patients carried homozygous truncating variants (n=6) and a homozygous missense variant (n=1) with variants located in domains D2, D3 or D4. However, the limited allelic diversity in this cohort, which is dominated by a recurrent founder variant and may be related to the shared ethnic background of many individuals, and the variability in survival among individuals carrying the same variant indicates that factors beyond variant location contribute to clinical outcome. These may include genetic background, consanguinity, family history of SWS, access to specialized medical care, and regional differences in healthcare. Although our functional data suggest more severe consequences of LIFR variants in the cytoplasmic region, domain location alone should not be considered a reliable predictor of survival. Of note, 73% of individuals in our cohort were born to consanguineous parents, and the cohort presents a strong predominance of individuals of Arabic ethnicity that may present a bias. Further studies in more ethnically and geographically diverse cohorts will be required to validate these observations.

We focused on the homozygous missense variant p.Cys270Gly in the D3 domain, found in a patient with several signs of classic Stüve-Wiedemann syndrome who survived infancy but succumbed to COVID-19-MIS-C at the age of six years and eight months. To confirm the pathogenicity of this variant, our work in Ba/F3 cells with this mutation confirmed (i) reduced cellular proliferation and phosphorylation of STAT3 and ERK after treatment with LIF, (ii) absent phosphorylation of STAT1 after treatment with LIF, and (iii) reduced LIFR cell surface expression by flow cytometry indicating a lower surface presentation of the LIFR p.Cys270Gly variant compared to wild type. As total LIFR expression in cells was similar to wildtype, we noted that this variant leads to altered LIFR protein localization from the cellular surface to the cytoplasm by endocytosis, ultimately prompting lysosomal degradation for recycling. Mechanistically, endocytosis and lysosomal degradation of LIFR are mediated by the adaptor protein AP2 and the kinase ERK (47). The proinflammatory cytokine tumor necrosis factor alpha (TNFa) downregulates LIFR expression and injection of lysosomal inhibitors including chloroquine rescue LIFR expression (48). We present the first study that shows reduced ERK activity in human LIFR deficiency. While previous studies have shown reduced STAT3 phosphorylation with LIFR-SWS (2, 20, 49), only studies in mouse and cellular models have previously examined the effect on ERK (50, 51). LIFR loss-of-function in SWS may provide a distinct pathomechanism including dysregulated endolysosomal trafficking that is particularly relevant in the survival of post-mitotic tissue where cellular survival hinges upon degradation of dysfunctional cargo for protein quality control (52). We delineated phenotypes with core manifestations in the nervous system, the skeleton, and immune cells, which are marked by a rather high degree of post-mitotic cells for which protein and organellar quality control mechanisms are essential for viability. Of note, there is a distinct phenotypic overlap in patients with Stüve-Wiedemann syndrome and lysosomal or autophagy disorders, particularly with neurodevelopmental disorders, neuroinflammation, and short stature (53). Recent reports on defects in autophagolysosomal tethering with EPG5-related Vici syndrome with mitochondrial myopathy and neurodegenerative disease courses show an upregulation in STING in neuronal cells (54). Notably, we also found upregulation in TMEM173/STING in LIFR:p.Cys270Gly bronchoalveolar cells without a presence of an infection, supporting the notion of a chronic hyperinflammation in bronchoalveolar cells.

Loss of LIFR was previously reported to activate NF-κB signaling (55), eventually prompting STING activation. Our clinical data on milder LIFR-disorders (Table 1) show survival beyond infancy but myopathic and neurodegenerative disorders later in childhood, further aided by two reports of mitochondrial myopathy in SWS patients (35) and by neurodegeneration in histopathological analyses of Lifr knockout mice (56), while previous reports on truncating variants showed demise at infancy (15, 20, 32).

To gain additional insight into dysregulated endolysosomal trafficking, we chose the p.Cys270Gly variant for our proteomics analyses in patient bronchoalveolar cells. First analyses on key proteins involved in endolysosomal trafficking showed upregulations of the AP2 subunits AP2A1 and AP2A2 (Figure 4) indicative of increased endocytosis trafficking in patient bronchoalveolar cells, in line with the previous observation of AP2-mediated endosomal trafficking of LIFR (47). Another notable finding in this pathway was upregulation of the endosome-associated adaptor protein APPL1, a Rab5 effector previously identified in endosomal trafficking of LIFR (12). In line with this, we observed an upregulation in endosomal proteins (RAB5A/C) involved in maturation of early endosomes to lysosomes. This proteomic signature is further indicated by the upregulation of proteins associated with trafficking from ER to Golgi (RAB1A/B-RAB6A), trafficking from Golgi to early endosomes (RAB31-AP3D1-GABARAP), trafficking of lipid droplets (RAB18), and trafficking from plasma membrane to endosomes (RAB10-RAB13). This proteomic signature of endosomal dysregulation demonstrates an attempt to internalize dysfunctional LIFR for recycling. However, it shall be noted that this exploratory finding in the proteomic analysis of bronchoalveolar lavage (BAL) cells was performed in samples obtained from one patient and one control subject, and further proteomic analyses in larger cohorts are required in follow-up investigations.

Whereas, the notion of dysregulated endosomal trafficking response further extends to RAB35, involved in xenophagy and mitophagy (57), as well as RAB7A, VPS11 and LAMP1, involved in autophagolysosomal fusion (58), and notably, downregulation of cathepsin L. At physiological conditions, activated STAT3 upregulates cathepsin L, which enhances lysosomal membrane permeabilization for adequate autophagolysosomal flux (59). In a murine retinal degeneration model, inhibition of STAT3 signaling with niclosamide led to stalled autophagolysosomes (60). Notably, STAT3 phosphorylation leads to BNIP3 activation, a potent mitophagy regulator (61), raising the discussion whether, in turn, defective mitophagy may contribute to further waste accumulation. Upon investigating late endosomal proteins, we observed downregulation of CD63 and upregulation of its negative regulator CD9, both involved in endosomal fusion with the plasma membrane to ultimately release vesicles into extracellular space (62). STAT3 failed to localize to late endosomes in STAT3-mutant mice (63). In line with this, we have shown attenuated STAT3 activation upon LIF stimulation in p.Cys270Gly BaF/3 cells. Downregulation of exocytosis in light of reduced STAT3 activation may be particularly important for neuronal health in patients, in whom neurodegenerative manifestations include epileptic encephalopathy and spastic paraplegia (Table 1), similar to TBCK-related neurodevelopmental disorders where short stature with upregulations of RAB5 (64) indicate a common pathway to compensate for accumulation of intracellular waste.

Moreover, short stature remains a notable phenotype in the context of reduced STAT3 activation, as STAT3 deficiency (Job’s syndrome) is associated with similar skeletal anomalies as SWS, but usually STAT3 gain-of-function mutations are observed with short stature. Notable findings involved in growth-related proteins from our proteomics assays included downregulations in IGFBP3, IGFBP1, and IGF2. GO molecular function term analyses indicated upregulation of catabolic processes for keratan sulfate, glycosaminoglycans, and aminoglycans which are primarily involved in cartilage and the extracellular bone matrix. In addition, a mouse model of STAT3 deficiency shows bowing of long bones and short stature (65), perhaps analogous to the human SWS phenotype with reduced STAT3 phosphorylation in the above experiments for the p.Cys270Gly variant. The early spontaneous fractures and the hip cartilage defects in these STAT3-deficient mice may also be due to an increased catabolism of extracellular bone-matrix proteins. We discuss that short stature with bowing of long bones in SWS may be distinct from the short stature in STAT3 GOF.

Furthermore, the observed MIS-C in our patient with a biallelic p.Cys270Gly missense variant may serve as an important note for other patients with SWS. In a published report on COVID19-related patient plasma samples, LIFR expression correlated with significantly less hospitalization and lower progression to severe acute illness (66). IL6-receptor (IL-6R) antagonists such as tocilizumab have been used in the acute management of COVID19-related infection with variable efficacy, possibly accounted for by variants in the paralog gene IL6ST/gp130 (67). However, it is unclear whether tocilizumab would be beneficial in the setting of MIS-C with LIFR deficiency. As part of the life-threatening LIFR-related disorder, our clinical data indicate multiple bouts of infections including pneumonia (2), while intravenous immunoglobulin (IVIG) infusions were reported as beneficial with hypogammaglobulinemia in SWS type 2 individuals with variants in the paralog gene IL6ST (68, 69), and subcutaneous administration (SCIG) for IL6ST-related urticaria (70). While steroid treatment may also dampen hyperinflammatory processes with only anecdotal support, the monoclonal IgG4 antibody dupilumab was reported for IL6ST-related eczema (70). We also observed an upregulation in FCER1G, part of the high-affinity IgE receptor. Only IL6ST-related disorders were previously associated with elevated IgE levels (3). Triggering IgE receptors with anti-IgE-antibody led to a rapid release of LIF in human lung tissues (71). While it is conceivable that IgE elevation may be relevant in LIFR deficiency, there is no sufficient clinical data to correlate the human disease course with IgE elevation at this time.

Hyperthermia was presented mostly at infancy and early childhood in LIFR-related disease, and the life-threatening episodes were occasionally associated with an early death. We propose that patients should be monitored regularly by immunological services even after survival beyond infancy, in particular during intercurrent viral infections to prevent life-threatening events. In line with malignant hyperthermia in RYR1-associated neuromuscular disorders (72–74), the association of SWS with mitochondrial myopathy shall prompt that depolarizing medications are to be excluded in the care of patients with SWS (75), further highlighted by a report with rhabdomyolysis after anesthesia (76).

Figure 5 illustrates the expanding phenotype of LIFR-disorders showing the overlapping and distinctive features towards IL6ST-disorders. Taken together, our studies in a murine cellular model and LC-MS proteomic analyses of human samples gave detailed insight into dysregulated pathways of endolysosomal trafficking, immune response and growth-associated proteins. The expanded phenotypes in our novel 22 patients will aid in genetic counselling and surveillance of patients and families.

Acknowledgments

We thank Yvonne Arlt for technical and Petra Oprée for organization assistance.

Funding Statement

The author(s) declared that financial support was not received for this work and/or its publication.

Footnotes

Edited by: Francesco Saettini, Fondazione IRCCS San Gerardo dei Tintori, Italy

Reviewed by: Robert Sokolic, United States Food and Drug Administration, United States

Roukaya Yaakoubi, University of Rochester, United States

Data availability statement

The original contributions presented in the study are included in the article/Supplementary Material; further inquiries can be directed to the corresponding author/s. The raw proliferation data supporting the conclusions of this article are available from the authors upon reasonable request. Restrictions apply to the proteomics datasets: the datasets are not readily available because they were generated from patient samples and cannot be publicly shared due to privacy and re-identification concerns.

Ethics statement

The studies involving humans were approved by 20-1711, Medical Faculty of University Cologne, Germany. The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent for participation in this study was provided by the participants’ legal guardians/next of kin.

Author contributions

MS: Writing – original draft, Writing – review & editing, Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Resources, Validation, Visualization. PR: Writing – original draft, Writing – review & editing, Data curation, Formal analysis, Investigation, Methodology, Resources, Validation, Visualization. ASl: Writing – review & editing, Data curation, Investigation, Resources. GZ: Writing – review & editing, Resources, Project administration. MW: Writing – review & editing, Formal analysis, Investigation. JDS: Writing – review & editing. SK: Writing – review & editing. RK: Writing – review & editing. CV: Writing – review & editing. CN: Writing – review & editing. OS: Writing – review & editing. MA: Resources, Writing – review & editing, Investigation. HA: Resources, Writing – review & editing, Investigation. ES: Resources, Writing – review & editing, Investigation. SJ: Resources, Writing – review & editing, Investigation. BS: Resources, Writing – review & editing, Investigation. NK: Resources, Writing – review & editing, Investigation. PB: Resources, Writing – review & editing, Investigation. SN: Resources, Writing – review & editing, Investigation. RP: Resources, Writing – review & editing, Investigation. KG: Resources, Writing – review & editing, Investigation. AH: Resources, Writing – review & editing, Investigation. AC: Resources, Writing – review & editing, Investigation. RH: Resources, Writing – review & editing, Investigation. KH: Resources, Writing – review & editing, Investigation. ID: Resources, Writing – review & editing, Investigation. JK-R: Writing – review & editing. GD: Writing – review & editing. RE: Resources, Writing – review & editing, Investigation. MK: Resources, Writing – review & editing, Investigation. TN: Writing – review & editing. VB: Writing – review & editing. HJ: Writing – review & editing. AA: Writing – review & editing, Supervision. JS: Writing – review & editing, Supervision, Data curation, Investigation, Methodology, Project administration, Resources, Validation. HD: Writing – review & editing, Writing – original draft, Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Visualization. KA: Writing – review & editing, Investigation, Data curation. AMK: Writing – review & editing, Investigation, Data curation. PB: Writing – review & editing, Resources, Project administration.

Conflict of interest

Author GZ was employed by the company CENTOGENE GmbH. Authors RP, KG were employed by the company Suma Genomics Private Limited.

The remaining author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Generative AI statement

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Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu.2026.1927542/full#supplementary-material

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Associated Data

Supplementary Materials

Data Availability Statement

The original contributions presented in the study are included in the article/Supplementary Material; further inquiries can be directed to the corresponding author/s. The raw proliferation data supporting the conclusions of this article are available from the authors upon reasonable request. Restrictions apply to the proteomics datasets: the datasets are not readily available because they were generated from patient samples and cannot be publicly shared due to privacy and re-identification concerns.