Deciphering genetic uncertainty in familial HLH

In this issue of Blood, Noori et al describe a novel in vitro approach to model germline variants in genes associated with familial hemophagocytic lymphohistiocytosis (fHLH). This approach allows for analysis of variants of uncertain significance (VUS), shedding light on the functional consequences of these variants.¹ 

fHLH, also known as primary HLH, comprises a group of rare hyperinflammatory disorders characterized by germline biallelic loss-of-function (LOF) mutations in the genes PRF1, UNC13D, STXBP2, or STX11, each of which is required for CD8 T cell– and natural killer (NK) cell–mediated cytotoxicity. In patients with fHLH, CD8 T and NK cells become activated following little to no immune trigger and are unable to eradicate targets, such as pathogen-infected cells and activated antigen-presenting cells. As a consequence, patients with fHLH develop exuberant immune cell activation with release of proinflammatory cytokines that mediate fatal multiorgan failure if untreated.² Although medications that induce effector cell apoptosis or inhibit cytokine activity can temporarily control hyperinflammation, definitive treatment requires stem cell transplantation.³ 

Optimizing outcomes in fHLH relies on making a rapid and accurate diagnosis, which currently necessitates identification of LOF mutations in 1 of the fHLH genes. fHLH is associated with genetic diversity, and sequencing frequently identifies novel, rare, or even relatively common VUS, which contribute little to clinical decision making.⁴ To facilitate interpretation of sequencing results, several assays have been developed to evaluate how germline variants impact cell function. One such assay measures the translocation of CD107a (also known as LAMP-1), a protein contained within cytotoxic granules, to the surface of patient-derived effector cells following stimulation, serving as a surrogate for degranulation. Although decreased CD107a expression can accurately identify patients with biallelic mutations in UNC13D, STX11, and STXBP2, CD107a expression is normal in cells from patients harboring LOF mutations in PRF1.⁵ A complementary assay exists to examine the ability of patient-derived NK cells to kill target cells in vitro. This assay, however, is subject to poor specificity because hyperinflammation can suppress NK cell killing,⁶ resulting in an abnormal result even in the absence of LOF mutations. The performance of both assays depends on obtaining sufficient numbers of CD8 T or NK cells, which are often reduced in patients with active HLH. Thus, it can be challenging to carry out these assays and interpret their results.⁷ 

The genetic complexity of fHLH and the caveats of existing functional assays pose diagnostic challenges. To address this gap, Noori et al developed a novel method for functional analysis of variants identified in patients suspected of having fHLH that uses mouse CD8 T cells. Their approach starts with CRISPR/Cas9-mediated knockout of the endogenous mouse gene of interest (Prf1, Unc13d, Stx11, or Stxbp2), which results in impaired CD107a translocation and/or cytotoxicity. They then express a complementary DNA (cDNA) of the corresponding human gene engineered to contain a patient-derived mutation. Using flow cytometry to measure CD107a upregulation and a chromium release assay to assess lymphocyte cytotoxicity,⁸ they evaluate whether expression of the mutant cDNA rescues the functional defects conferred by gene knockout (see figure). The authors validated this approach using germline variants identified in patients meeting clinical criteria for HLH. Intriguingly, when they modeled variants from patients who presented before age 5 years, none of the variants produced detectable cytotoxic activity. In contrast, many variants from patients presenting later in life partially rescued cytotoxicity, suggesting that residual gene function protected against early development of disease. Thus, the quantitative readouts of their assay appear to correlate with HLH clinical presentation.

The in vitro assay developed by Noori et al is notable in that it enables the functional evaluation of novel or uncertain genetic variants in fHLH genes, holding potential to facilitate interpretation of variant pathogenicity and inform treatment decisions. Nevertheless, there are several questions and caveats that remain. For example, can this system be effectively translated to the clinical setting? The in vitro assay requires generation of mutant cDNA expression vectors and genetic manipulation of mouse CD8 T cells. The assay, therefore, requires time and technical expertise. It is also vulnerable to incomplete knockdown of the endogenous mouse fHLH genes and variable expression of the corresponding human proteins. Results must be carefully interpreted in the context of these potential limitations. Further, this assay is designed to investigate the functional consequences of a single fHLH gene variant. However, patients presenting with HLH commonly harbor compound heterozygous variants (affecting the same or different fHLH genes), with the combined effect of the 2 mutant proteins impairing cytotoxicity. Rarely, patients may also inherit a single fHLH gene variant whose encoded protein exerts a dominant-negative effect on the wild-type protein.⁹ Neither of these effects is easily studied using the current system, in which single variants are expressed in isolation. Surprisingly, when establishing their assay, the authors discovered that knockdown of fHLH genes in human CD8 T cells does not lead to degranulation or cytotoxic defects, yet it does so in mouse cells. Conversely, addition of interleukin-2 to cultures does not restore the functions of Stx11- or Stxbp2-deficient mouse CD8 T cells, but it does so in human cells. These findings highlight the biologic differences between species and remind us “mice are not little humans.” Thus, whereas the results of the functional assay appear to correlate with human clinical features, more needs to be done to understand these differences and confirm that the readouts of the current assay, which uses mouse CD8 T cells, fully translate to the human setting.

Clinically, fHLH shares features with other inflammatory conditions, including sepsis and systemic inflammatory response syndrome.¹⁰ Accurate diagnosis is crucial to clinical decision making, as immunosuppressive therapies and stem cell transplantation are contraindicated in these other conditions. Although genetic diagnosis remains the gold standard, understanding the consequences of patient-derived germline variants has been a barrier to interpreting clinical sequencing results. The work by Noori et al paves the way for functional interrogation of these variants, allowing for an assessment of pathogenicity that can facilitate diagnosis. The authors are commended for their development of this novel assay; however, prospective validation of this approach is needed to further evaluate its feasibility and allow for the development of standardized workflows for routine clinical implementation.

Conflict-of-interest disclosure: The authors declare no competing financial interests.