Background Growing evidence reveals a pathological role of somatic mutations in various autoimmune diseases, such as the mutation in UBA1 in VEXAS syndrome, CARD11 and KLHL6 in cryoglobulinemic vasculitis, or STAT3 in Felty’s syndrome^[1]. Somatic mutations might also be involved in the pathogenesis of ANCA-associated vasculitis (AAV), which typically manifests in middle-aged and elderly individuals.

Objectives We aimed to identify somatic mutations in patients with AAV.

Methods We collected whole blood and obtained peripheral blood mononuclear cells (PBMCs) and neutrophils from patients with AAV in active-disease status (n=16), as well as from patients with other autoimmune diseases (n=8) as disease controls, and healthy subjects (n=10). In addition, we collected these specimens from 12 out of the 16 patients with AAV after remission induction. We performed RNA sequencing (RNA-seq) on the obtained cells and whole genome sequencing (WGS) on DNA extracted from the whole blood. Somatic mutations were detected by comparing the RNA and DNA sequences^[2].

Results After stringent quality control, we identified 108 somatic mutations across 16 patients in active-disease status. The mean coverage of RNA-Seq at the mutation site was 100.9 ± 367.8 ×, and that of WGS was 14.4 ± 4.3 ×, while the mean allele fraction was 22.9 ± 20.5%. One or more mutations were detected in each of the 15 (93.8%) patients. The median mutation count of each patient was 4.0, which was not significantly different from disease controls or samples after remission induction. We mapped one gene to each of the 108 mutations, resulting in 95 genes in total. Mutations for six of the 95 genes were observed in two or more patients, and two of them were related to the ubiquitin system. Of the 108 mutations, 37 were missense, and 20 were predicted to be deleterious (combined annotation-dependent depletion Phred score > 20). Among the 20 mutations, the HIST2H2AC mutation (NM_003517: p.L86P) in neutrophils was observed in two patients. To evaluate the functional outcome of the 20 mutations, we analysed the data on knocking out the corresponding genes using CRISPR in K562 cells^[3]. The results showed that the expression of genes related to antigen presentation was increased in HEATR1 and RPL18 knockouts. When we followed up with 12 of the 16 patients after remission induction, 81.4% of the somatic mutations were no longer detected. Additionally, 91.7% of the deleterious mutations disappeared.

Conclusion We found somatic mutations with potential pathological effects in some patients with AAV exhibiting active-disease status. Notably, the majority of these mutations were not detected after remission induction.

References

1. Mustjoki, S. & Young, N. S. Somatic Mutations in ‘Benign’ Disease. N. Engl. J. Med. 384, 2039–2052 (2021).
2. Yizhak, K. et al. RNA sequence analysis reveals macroscopic somatic clonal expansion across normal tissues. Science 364, (2019).
3. Replogle, J. M. et al. Mapping information-rich genotype-phenotype landscapes with genome-scale Perturb-seq. Cell 185, 2559-2575.e28 (2022).

Acknowledgements The present study was supported in part by JSPS KAKENHI. (Grant No. 19H03209).

Disclosure of Interests None Declared.

Keywords: Vasculitis, Genetics/Epigenetics

DOI: 10.1136/annrheumdis-2023-eular.243