Targeted high-throughput sequencing for genetic diagnostics of hemophagocytic lymphohistiocytosis

Abstract

BackgroundHemophagocytic lymphohistiocytosis (HLH) is a rapid-onset, potentially fatal hyperinflammatory syndrome. A prompt molecular diagnosis is crucial for appropriate clinical management. Here, we validated and prospectively evaluated a targeted high-throughput sequencing approach for HLH diagnostics.MethodsA high-throughput sequencing strategy of 12 genes linked to HLH was validated in 13 patients with previously identified HLH-associated mutations and prospectively evaluated in 58 HLH patients. Moreover, 2504 healthy individuals from the 1000 Genomes project were analyzed in silico for variants in the same genes.ResultsAnalyses revealed a mutation detection sensitivity of 97.3 %, an average coverage per gene of 98.0 %, and adequate coverage over 98.6 % of sites previously reported as mutated in these genes. In the prospective cohort, we achieved a diagnosis in 22 out of 58 patients (38 %). Genetically undiagnosed HLH patients had a later age at onset and manifested higher frequencies of known secondary HLH triggers. Rare, putatively pathogenic monoallelic variants were identified in nine patients. However, such monoallelic variants were not enriched compared with healthy individuals.ConclusionsWe have established a comprehensive high-throughput platform for genetic screening of patients with HLH. Almost all cases with reduced natural killer cell function received a diagnosis, but the majority of the prospective cases remain genetically unexplained, highlighting genetic heterogeneity and environmental impact within HLH. Moreover, in silico analyses of the genetic variation affecting HLH-related genes in the general population suggest caution with respect to interpreting causality between monoallelic mutations and HLH. A complete understanding of the genetic susceptibility to HLH thus requires further in-depth investigations, including genome sequencing and detailed immunological characterization.Electronic supplementary materialThe online version of this article (doi:10.1186/s13073-015-0244-1) contains supplementary material, which is available to authorized users.

Advances in the pathogenesis of primary and secondary haemophagocytic lymphohistiocytosis: differences and similarities

“…Presumably they are not clinically neutral given their enrichment in patients with secondary HLH compared to healthy or inflammatory controls (Zhang et al, 2008a;Vastert et al, 2010;Kaufman et al, 2014). Only one study reported no enrichment of monoallelic rare variants in secondary HLH, compared to a large healthy control cohort (Tesi et al, 2015b). Theoretically, hypomorphic single-copy mutations may alter protein levels and thus induce subtle impairment of cytotoxicity, increasing susceptibility to infections.…”

“…Theoretically, hypomorphic single-copy mutations may alter protein levels and thus induce subtle impairment of cytotoxicity, increasing susceptibility to infections. Interestingly, heterozygous mutations in PRF1 and LYST have also been implicated in severe H1N1 influenza (Schulert et al, 2016), suggesting the sequence variants are not strictly recessive but may generate haploinsufficiency for cytotoxicity genes (Zhang et al, , 2014aTesi et al, 2015b;Cetica et al, 2016).…”

Polygenic mutations in the cytotoxicity pathway increase susceptibility to develop HLH immunopathology in mice

“…30,40 Besides, there is no evidence of an increase in rare, putatively pathogenic monoallelic variants in HLH-associated genes in secondary HLH patients so far analyzed. 41 Prospective studies analyzing the different HLH-related genes systematically and the combination of the genes' variants in large cohorts of secondary HLH patients, should help in evaluating the contribution of this genetic mechanism in conferring a greater risk in developing HLH.…”

Hermansky–Pudlak syndrome: Mutation update

“…Next‐generation sequencing of different cohorts of subjects with primary immunodeficiency or hemophagocytic lymphohistiocytosis (HLH) reported least eight AP3B1 variants (Table S4) in a heterozygous state with or without a variant in another (synergistic) gene. These findings suggest that heterozygous AP3B1 variants may contribute to an immunologic phenotype (Chi et al, 2018; Gallo et al, 2016; Gao, Zhu, Huang, & Zhou, 2015; Miao et al, 2019; Mukda et al, 2017; Tesi et al, 2015; Xu et al, 2017). These variants have not been reported in HPS subjects but were included in this report because they may cause HPS when occurring in a homozygous or compound heterozygous state.…”