Date: Monday, November 8, 2021
Session Title: Pediatric Rheumatology – Basic Science Poster (1007–1013)
Session Type: Poster Session C
Session Time: 8:30AM-10:30AM
Background/Purpose: Splice site variants in IKBKG that lead to exon 5 deletion cause NEMO-deleted exon 5 autoinflammatory syndrome (NEMO-NDAS). NEMO-NDAS clinically mimics the interferonopathy chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature (CANDLE) but treatment with JAK inhibitors provides only partial benefit. Because an IKBKG pseudogene complicates the genetic diagnosis of NEMO-NDAS by standard methods, we developed and validated a bioinformatics approach to diagnose these patients (pts). We further characterized the complex immunodysregulation present in NEMO-NDAS.
Methods: A bioinformatics pipeline to mask the IKBKG pseudogene was refined using splice prediction tools (SpliceAi, TraP and MaxEntScan). Screening of IKBKG exon 5 ± 30bp was validated in 701 samples from subjects enrolled in an IRB-approved protocol, and in internal (n=2655, whole exome sequencing (WES)) and public (n=2498, whole genome sequencing (WGS)) databases. The variants detected were validated by Sanger or amplicon deep sequencing (seq) and spliced product was confirmed by Western blot, cDNA seq and RNA-seq. Nanostring gene expression, PBMC stimulation and cytokine assays were performed. CRISPR generated U937 cell line clones were functionally assessed.
Results: 13 pts (9 females and 4 males) had 9 different de novo splice site variants in IKBKG. cDNA seq (12/12) or Western blot (5/5) confirmed the splice product in all pts tested. RNA-seq (n=12) showed a high frequency of exon 5 skipping (median 55% (35-80%)). IKBKG exon 5 splice site variants were screened in internal and public WES/WGS databases (n=5149) and 11 variants in 22 subjects passed filters. Sanger seq confirmed 1 of the 11 variants (9%); amplicon deep seq is pending. The most common clinical features in NEMO-NDAS pts were panniculitis with systemic inflammation (100%), ectodermal dysplasia (83%), hepatosplenomegaly (77%) and B-cell lymphopenia (80%). Compared to CANDLE pts (n=5), NEMO-NDAS pts’ skin biopsies (n=7) showed histiocytic panniculitis, vacuolar interface changes and dyskeratosis. Liver biopsies (n=3) showed granulomatous hepatitis; 2 other pts had portal hypertension. All pts were steroid-dependent with partial responses to anti-TNF (n=9) or JAK inhibition (n=9). Nanostring IFN and NF-κB scores were elevated in all 13 pts. Pts with NEMO-NDAS had higher serum levels of IFN-γ, IL-12p40, IL-17 and IL-23 than seen in CANDLE pts (p< 0.0001 for all). Stimulated M1 and M2 macrophages from NEMO-NDAS pts (n=3) produced higher levels of CCL3/4 (MIP1-α/β) than healthy control (HC) cells (n=7)(p< 0.05). NEMO-NDAS pts (n=2) had normal T and B cell proliferation, and Fas-induced T cell death was comparable to HC. U937 cell line clones lacking exon 5 normally degraded IκBα upon LPS stimulation and mutant U937 clones showed enhanced TNF induced cell death compared to wildtype and PSMB8-/- clones.
Conclusion: Our bioinformatics pipeline masking IKBKG pseudogene provides a sensitive diagnostic tool for the early recognition of NEMO-NDAS pts. The role of cytokine dysregulation and TNF induced cell death in the specific pathogenesis of NEMO-NDAS is being evaluated to improve therapeutic options.
This work was supported by the NIH IRP of NIAID
To cite this abstract in AMA style:Almeida de Jesus A, Lin B, Karlins E, Kahle D, Rastegar A, Mitchell J, Torreggiani S, Bhuyan F, Alehashemi S, Cetin Gedik K, Uss K, Lee C, Kuehn H, Rosenzweig S, Calvo K, Walkiewicz M, Lack J, Hanson E, Khojah A, Wu E, Scott C, Ronan Leahy T, MacDermott E, Kileen O, Arkachaisri T, Gucev Z, Cook K, Mammadova V, Nasrullayeva G, Canna S, Kuhns D, Dalgard C, Moran T, Oler A, Goldbach-Mansky R. Validation of Bioinformatics Pipeline to Detect NEMO-Deleted Exon 5 Autoinflammatory Syndrome (NEMO-NDAS) and Preliminary Clinical and Immunologic Characterization [abstract]. Arthritis Rheumatol. 2021; 73 (suppl 9). https://acrabstracts.org/abstract/validation-of-bioinformatics-pipeline-to-detect-nemo-deleted-exon-5-autoinflammatory-syndrome-nemo-ndas-and-preliminary-clinical-and-immunologic-characterization/. Accessed January 27, 2023.
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