Gain-of-function mutations in ALPK1 cause an NF-κB-mediated autoinflammatory disease: functional assessment, clinical phenotyping and disease course of patients with ROSAH syndrome

Objectives To test the hypothesis that ROSAH (retinal dystrophy, optic nerve oedema, splenomegaly, anhidrosis and headache) syndrome, caused by dominant mutation in ALPK1, is an autoinflammatory disease. Methods This cohort study systematically evaluated 27 patients with ROSAH syndrome for inflammatory features and investigated the effect of ALPK1 mutations on immune signalling. Clinical, immunologic and radiographical examinations were performed, and 10 patients were empirically initiated on anticytokine therapy and monitored. Exome sequencing was used to identify a new pathogenic variant. Cytokine profiling, transcriptomics, immunoblotting and knock-in mice were used to assess the impact of ALPK1 mutations on protein function and immune signalling. Results The majority of the cohort carried the p.Thr237Met mutation but we also identified a new ROSAH-associated mutation, p.Tyr254Cys. Nearly all patients exhibited at least one feature consistent with inflammation including recurrent fever, headaches with meningeal enhancement and premature basal ganglia/brainstem mineralisation on MRI, deforming arthritis and AA amyloidosis. However, there was significant phenotypic variation, even within families and some adults lacked functional visual deficits. While anti-TNF and anti-IL-1 therapies suppressed systemic inflammation and improved quality of life, anti-IL-6 (tocilizumab) was the only anticytokine therapy that improved intraocular inflammation (two of two patients). Patients’ primary samples and in vitro assays with mutated ALPK1 constructs showed immune activation with increased NF-κB signalling, STAT1 phosphorylation and interferon gene expression signature. Knock-in mice with the Alpk1 T237M mutation exhibited subclinical inflammation. Clinical features not conventionally attributed to inflammation were also common in the cohort and included short dental roots, enamel defects and decreased salivary flow. Conclusion ROSAH syndrome is an autoinflammatory disease caused by gain-of-function mutations in ALPK1 and some features of disease are amenable to immunomodulatory therapy.

reporter under a NF-κB-responsive element, a firefly expression vector, and an ALPK1 cDNA construct carrying either a WT or mutant sequence. Twenty-four hours after transfection, luciferase activity was measured using the Nano-Glo® Luciferase Assay System (Promega), and the NanoLuc® luciferase activity was normalized against the firefly luciferase activity for control of transfection efficiency and cell number. The reporter activity was then normalized to the result of WT construct.
Cell stimulation and immunoblotting ADP-heptose (Invivogen) was used to stimulate fibroblast and transfected 293T cells (5-10 uM) for indicated times. Whole cell lysates were prepared using ice-cold 1x cell lysis buffer (Cell Signaling) supplemented with complete protease inhibitors. Immunoblotting was conducted using specific antibodies as described previously. ImageJ was used to analyze the immunoblotting images.
In vitro pSTAT1 phosphorylation assay PBMCs from ROSAH patients and healthy donors were isolated by Ficoll (Ficoll-Paque PLUS; GE Healthcare) density-gradient centrifugation. STAT1 phosphorylations was assessed by treating PBMCs with IFN-α or IFN-γ (Cell Signaling Technology, Boston, MA) (200ng/ml) at 37°C for 20 minutes. The stimulation was stopped by addition of 1X Lyse/Fix Buffer (BD Biosciences, San Diego, CA). The PBMCs were then washed with PBS and permeabilized in ice-cold Phosflow Perm Buffer III (BD Biosciences, Sand Diego, CA) for 30 minutes in the dark. Cells were washed with FACS buffer (PBS, pH 7.4, 1% BSA, 4 mM EDTA, 0.2% NaN 3 ). Monoclonal antibodies to CD3, CD4, CD8, and CD14 (BD Biosciences, San Diego, CA) were used to identify CD4 + T cells, CD8 + T cells and monocytes respectively. STAT1 phosphorylations were evaluated with 20 mL of Alexa Fluor647 pSTAT1 antibody (BD Biosciences, San Diego, CA). Flow cytometric analysis was performed on a FACS Fortessa (BD Immunocytometry, San Jose, CA) with Diva software. Data were analyzed using FlowJo software (Treestar, Ashland, OR). Median fluorescence intensity (MFI) of the corresponding pSTAT1 were calculated for each cell subset.

Mice
All animal studies were performed in accordance with guidelines from Ministry of Health, China or the National Institutes of Health and were approved by the Institutional Animal Care and Use Committee of National Institute of Biological Sciences, Beijing or National Human Genome Research Institute, Bethesda, MD or National Eye Institute, Bethesda, MD, respectively. To generate Alpk1 T237M/ T237M mice, gRNA (ACAGGGCATTTCCACATCAC) targeting exon 9 of Alpk1 and donor atgagagaattccagtctgatttgtttgtttttcctgacagggcatttccATGAGTTTAggcatactggcagacatctttgtttccatgag caaaaccgattatgaaaa were used. In vitro-transcribed guide RNA, Cas9 mRNA and donor were co-microinjected into C57BL/6N-derived zygotes. The tail genomic DNA of each offspring was amplified with the forward primer 5'-AGCCCAACTTCAAAGTAGCC -3' and the reverse primer 5'-CATCGAGAGAGGCTGGGATA -3'. Sanger sequencing was performed to analyze the PCR products and identify the founders with T237M mutation. Founders with the same mutation were intercrossed to obtain homozygous Alpk1 T237M mice.
Because C57BL/6N mice harbor an rd8 mutation in the Crb1 gene that results in a form of retinal degeneration, the C57BL/6N Alpk1 T237M mice were crossed with C57BL/6J mice that lacked the rd8 mutation.(8) Eight Alpk1 T237M/T237M Crb1 rd8/rd8 mice underwent ophthalmologic evaluation with fundus imaging and histologic evaluation of eyes from sacrificed mice. Three of the mice were imaged and sacrificed at 10 months of age and five of the mice were imaged and sacrificed at 7 months of age.
Five Alpk1 T237M/WT Crb1 rd8/WT mice were evaluated by ERG at 9 and 12 months of age. At 12 months of age, the mice were also evaluated for retinal degeneration by fundus imaging and optical coherence tomography (OCT) and Optodrum was used to assess visual acuity, as previously published. (9,10) Four Alpk1 T237M/WT Crb1 WT/WT mice were evaluated by ERG at 6 and 9 months of age. At 9 months of age, the mice were also evaluated for retinal degeneration by fundus imaging and OCT and Optodrum was used to assess visual acuity. Four wild-type control mice were evaluated by ERG at 6 months of age, but one died at 8 months of age so only 3 control mice were evaluated at 9 months of age.

Statistical analysis
Continuous variables are presented as means with standard deviations or medians with interquartile ranges and were compared with the use of parametric tests as appropriate.      A. Panel of dental panoramic X-rays from ROSAH individuals. Short roots are noted in all except F2.2. Multiple dental restorations present in F2.2 and F5.2, possibly due to enamel defects or caries. Multiple dental implants in F7.1. *marks teeth with taurodontism. +marks decayed or restored teeth.       . 1 1 1 . 1 1 1 . 2 1 1 . 3 1 2 . 1 1 3 . 1 1 1 . 1 1 1 . 2 1 1 . 3 1 2 . 1 1 3     Stimulated ROSAH leukocytes did not display increased intracellular cytokines (n=2). Whole blood cells from patient F1.1 and F2.4 were incubated with Phorbol 12-Myristate 13 Acetate and Ionomycin for 4 hours at 37°C to stimulate cytokine production. Activation was carried out in the presence of Brefeldin A, to inhibit intracellular transport, causing all cytokines produced during the activation to be retained inside the cell. The activated cells were stained with surface monoclonal antibodies for phenotyping, fixed, permeabilized and stained with antibodies to the cytokines IFN-γ, TNF-α and IL-4. The cells were then analyzed by flow cytometry. Percent positive results for IFN-γ, TNF-α and IL-4 were reported for CD4, CD8, NK and NKT cells.     Figure 10: NF-B signature.
Heatmap showing differentially expressed NF-B response genes (GO:0007249) in whole blood of pre-treatment (n=4) patients with ROSAH syndrome and healthy volunteers (n=3). Upregulated genes are shown in red, and down-regulated genes in blue. For patients F1.1 and F3.3, samples were collected on days for which sera and plasma cytokines were also significantly elevated, consistent with disease flare.  Table 8: List of the 99 differentially expressed inflammatory response genes (GO: 0006954) in whole blood of pre-treatment (n=4) and post-treatment with adalimumab (n=2) patients with ROSAH syndrome.