Background/Purpose: Macrophage activation syndrome (MAS) is a severe complication of rheumatic disease, particularly of systemic JIA (sJIA). It is currently classified among the secondary forms of HLH (sHLH). Primary HLH (pHLH) is caused by mutation of genes that code for proteins that are involved in cytotoxic functions. Mice carrying heterozygous mutations in more than 1 pHLH gene carry a higher risk to develop HLH following viral infection, suggesting that accumulation of partial genetic defects may be relevant in HLH.

Methods: Genes involved in pHLH were analysed, with next generation sequencing (NGS), in MAS in the context of different rheumatic diseases and in sHLH. A Targeted resequencing were performed on all patients using a panel including the 7 principal HLH-related genes (PRF1, UNC13d, STX11, STXBP2, RAB27a, XIAP, SH2D1A). Sequencing analysis were performed on the MiSeq® and NextSeq550® platforms (Illumina, San Diego, CA); all variants identified were confirmed by Sanger. The possible functional impact of variants was studied by in silico analysis using SIFT and PolyPhen softwares. We took into account only variants with anallelic frequency in the global population up to 1%, in the dbSNP and Ensembl databases, with the exception of A91V variant in PRF1 gene, because of its conflicting interpretation of pathogenicity.

Results: Fifty patients, 29 MAS (23 developed this complication in the context of sJIA, and 6 developed MAS respectively in the context of systemic vasculitis, Crohn’s disease, systemic lupus erythematosus, anti-phospholipid syndrome, chronic recurrent multifocal osteomyelitis and Kawasaki disease) and 21 patients with HLH secondary to infections or without any demonstrable trigger, were analysed. At least 1 heterozygous variant was identified in one of the pHLH-associated genes in 24 patients (12/29 MAS, 12/21 sHLH) with a detection rate of 48%. Seventeen patients (34%) showed only 1 variant, while more than 1 variant were found in 7 patients (14%). Seven (24%) patients with MAS showed the A91V variant in PRF1 in an heterozygous state, and 5 (17%) patients showed an heterozygous variant in UNC13d gene. Four of the 29 MAS patients had mutations in two different genes: one of them had recurrent episodes of MAS with a severe disease and a prolonged ICU admission. Two MAS patients carried 2 heterozygous variants in the same gene. Three (14%) patients with sHLH showed a heterozygous mutation in RAB27a, 3 in UNC13d and 4 in PRF1 gene. Two patients carried more than a single heterozygous variant: one had variants in two different genes, while one patient showed 3 different variants in the same gene (PRF1). For two of these patients with variants in two different genes, one of them presented three episodes of HLH reactivation and the other one presented a severe disease with exitus.

Conclusion: Mutations of PRF1 and UNC13d genes were frequently observed in patients with MAS or secondary HLH; RAB27a variants seems to be more frequent in patients with sHLH. Re-occurrence and severity of disease tend to be more frequent and more severe in sHLH patients who carry mutations in two genes. The contribution of these variants to the risk of MAS or sHLH remains to be established in the context of a polygenic model of sHLH and MAS.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/next-generation-sequencing-analysis-of-familial-haemophagocytic-lymphohistiocytosis-hlh-related-genes-in-macrophage-activation-syndrome-mas-and-secondary-hlh-shlh/