Identification of a patient with a novel ISG15 mutation. (A) Family includes two affected children carrying a homozygous variant (c.463insC) on ISG15 and six children of unknown status. The arrow indicates the patient described in this section (proband). (B) CT of the younger sibling taken at age 12. Extensive diffuse ground-glass opacities with subpleural sparing and enlargement of the main pulmonary artery are observed. (C) Schematic localization of the ISG15 variant in the genomic DNA locus indicated by the arrow. (D) Expression levels of various ISGs (IFI27, MX1, SIGLEC1, and IFIT1) were assessed from patient’s whole blood, as well as from the three healthy controls. Target genes were normalized on 18S rRNA expression. (E) 3′-RACE PCR targeting ISG15 was performed on RNA isolated from patient’s whole blood. (F) Sanger sequencing of the 3′-RACE band from E. (G) HEK293T cells were transfected with a plasmid encoding the ISG15 variant (c.463insC), ISG15 WT, or Luc. Relative mRNA levels for ISG15 were assessed by qRT-PCR, performed twice for each variant, with technical triplicates; the data from one representative experiment (n = 3) are shown. The expression level of ISG15 was normalized to GAPDH. (H) HEK293T cells were transfected with a plasmid encoding the ISG15 variant (c.463insC), ISG15 WT or Luc, plus HERC5, UBE1L, and UBCH8, as described in (23). Cell lysates were analyzed by western blotting for ISG15 and ISGylation; a representative experiment is shown. CT, chest tomography. Source data are available for this figure: SourceData F1.