Digenic and Multigenic Heterozygous FHL Genotypes Are Common but Clinically Silent in the General Population

Primary hemophagocytic lymphohistiocytosis is mainly caused by biallelic variants in genes disrupting cytotoxic NK- and T-cell function (PRF1, UNC13D, STX11, STXBP2, RAB27A, and LYST). A "pathway defect accumulation" model proposes that heterozygous variants in multiple FHL genes (digenic or multigenic inheritance) may increase susceptibility, but its significance remains debated. We assessed the prevalence and clinical relevance of di-/multigenic FHL genotypes in a German FHL cohort (1987-2023) and the UK Biobank (UKB, 469,589 participants). We analyzed (i) variants classified as disease mutations in the Human Gene Mutation Database (HGMD), (ii) common variants such as PRF1 p.Ala91Val and p.Asn252Ser as well as (iii) additional variants previously reported in digenic HLH and explored phenotypic associations using ICD-10 codes for possible HLH-related conditions. In the German cohort, among 635 individuals sequenced for more than one FHL gene, no symptomatic patient with abnormal NK/CTL-degranulation carried digenic/multigenic heterozygous variants. In UKB, 575 individuals carried digenic FHL genotypes (0.1% prevalence), without enrichment for HLH-associated phenotypes (OR=0.95; p=1). Four individuals carried trigenic genotypes; none had HLH-related diagnoses. Several HGMD-labeled pathogenic variants were observed biallelically in asymptomatic adults, suggesting potential misclassification. Including PRF1 p.Ala91Val and p.Asn252Ser increased digenic variant carriers to 2,590, but did not cause phenotype enrichment. Digenic heterozygous FHL variants are relatively common in the general population but do not confer increased FHL risk. Many reported pathogenic FHL variants may be benign. These findings argue against classifying multigenic heterozygous carriers as at risk and support integrating population data into variant interpretation.