Background/Purpose: The Inhibitor of Kappa-B Kinase Regulatory Subunit Gamma (IKBKG) is located on the X chromosome and encodes the NF-κB essential modulator (NEMO). Loss-of-function mutations in IKBKG cause immunodeficiency with ectodermal dysplasia in males and incontinentia pigmenti in females. We have recently reported 4 patients (pts) with gain-of-function (GOF) splice site variants in IKBKG causing an autoinflammatory disease (AID) that mimics chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature (CANDLE)¹. This AID has been called NEMO-deleted exon 5 autoinflammatory syndrome (NDAS). The presence of an IKBKG pseudogene (IKBKGP1) makes the genetic diagnosis of NEMO-associated diseases challenging. We sought to develop a customized bioinformatics approach as screening tool to facilitate the discovery of disease-causing splice site variants.

Methods: Pts enrolled in an IRB-approved protocol underwent whole exome/genome sequencing (WES/WGS) and trio analysis. A bioinformatics pipeline was developed to computationally mask the IKBKGP1 pseudogene and improve sensitivity of the discovery of splice site mutations. An interferon (IFN) response gene (IRG) score was assessed by Nanostring.

Results: Eight female pts with 5 different de novo splice-site variants in IKBKG were identified. The clinical phenotype of the 8 female pts was similar and, in some cases, more severe than the clinical phenotype of male pts with GOF IKBKG mutations previously reported. All female pts had early disease onset (2 days to 6 months-old) with nodular skin rashes, fever and increased inflammatory markers (ESR and CRP). Other frequent clinical manifestations included failure to thrive (8/8), lipodystrophy (4/8), hepatosplenomegaly (5/7), anemia (8/8), thrombocytopenia (6/8) and B-cell lymphopenia (5/7). All pts were cortico-dependent and partially responded to anti-TNF (n=5) or JAK inhibitor (n=5) therapies, and one pt deceased due to opportunistic infections (3 years-old). Analysis of pts’ WES/WGS data using a custom approach to mask IKBKG pseudogene revealed that two pts had variants in intron 5 (IKBKG NM_003639.4 c.671+5G >A in Pt1 and c.671+1G >A in Pt2); and 6 pts had variants in intron 4 (c.519-2A >G in Pt3-Pt6, c.519-22_519-14delGTCTGCTCT in Pt7 and c.519-7_519-6insGGCCCTGG in Pt8). None of the IKBKG variants had previously been detected by pts’ WES/WGS using standard analysis methods. Reverse transcription followed by cDNA sequencing showed exon 5 skipping in the 7 pts tested and Western blot confirmed the splice product in 2/2 pts. All 8 pts had high 28-gene IFN scores with a relatively higher expression of IRG’s that have transcription factor binding sites for NF-κB (CXCL10, GBP1 and SOCS1) and a high 3 NF-κB/25 IFN-gene ratio¹.

Conclusion: We describe de novo splice-site variants in the X chromosome gene, IKBKG (encoding NEMO), in 8 female pts with a CANDLE-like phenotype. Analysis of WES/WGS masking the IKBKG pseudogene provided a remarkably better diagnostic yield than standard methods, and could be used for the diagnosis of pts with NEMO-NDAS.
¹ de Jesus AA et al. J Clin Invest. 2020;130:1669-1682

Funding:  This work was supported by the Division of Intramural Research (DIR) of NIAID, NIH

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/splice-site-variants-in-ikbkg-encoding-nemo-detected-by-a-customized-analysis-of-next-generation-sequencing-data-cause-an-early-onset-autoinflammatory-syndrome-of-panniculitis-and-cytopenias-in-male/