A cell-based functional assay that accurately links genotype to phenotype in familial HLH

Blood. 2023 May 11;141(19):2330-2342. doi: 10.1182/blood.2022018398.


Familial forms of the severe immunoregulatory disease hemophagocytic lymphohistiocytosis (HLH) arise from biallelic mutations in the PRF1, UNC13D, STXBP2, and STX11 genes. Early and accurate diagnosis of the disease is important to determine the most appropriate treatment option, including potentially curative stem cell transplantation. The diagnosis of familial HLH (FHL) is traditionally based on finding biallelic mutations in patients with HLH symptoms and reduced natural killer (NK)-cell cytotoxicity. However, patients often have a low NK-cell count or receive immunosuppressive therapies that may render the NK-cell cytotoxicity assay unreliable. Furthermore, to fully understand the nature of a disease it is critical to directly assess the effect of mutations on cellular function; this will help to avoid instances in which carriers of innocuous mutations may be recommended for invasive procedures including transplantation. To overcome this diagnostic problem, we have developed a rapid and robust method that takes advantage of the functional equivalence of the human and mouse orthologues of PRF1, UNC13D, STX11, and STXBP2 proteins. By knocking out endogenous mouse genes in CD8+ T cells and simultaneously replacing them with their mutated human orthologues, we can accurately assess the effect of mutations on cell function. The wide dynamic range of this novel system allowed us to understand the basis of, otherwise cryptic, cases of FHL or HLH and, in some instances, to demonstrate that previously reported mutations are unlikely to cause FHL. This novel approach provides valuable new information to enable more accurate diagnosis and treatment of patients with HLH or FHL who inherit mutations of undetermined pathogenicity.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Genotype
  • Humans
  • Lymphohistiocytosis, Hemophagocytic* / diagnosis
  • Lymphohistiocytosis, Hemophagocytic* / genetics
  • Membrane Proteins / genetics
  • Mice
  • Munc18 Proteins / genetics
  • Mutation
  • Perforin / genetics
  • Phenotype
  • Pore Forming Cytotoxic Proteins


  • Pore Forming Cytotoxic Proteins
  • Perforin
  • UNC13D protein, human
  • Membrane Proteins
  • Stxbp2 protein, mouse
  • Munc18 Proteins