Discovery and functional analysis of a novel ALPK1 variant causing ROSAH syndrome

ROSAH syndrome is an autosomal dominant autoinflammatory disorder characterised by visual disturbance caused by pathogenic variation in the protein kinase ALPK1. Only two such variants have been reported to cause ROSAH syndrome to date: 66 out of 67 patients harbour the Thr237Met variant, while a single patient carries a Tyr254Cys variant. Here we identify a family in which ROSAH syndrome is caused by a Ser277Phe variant in ALPK1. The phenotypic variability in this family is high, with four of the seven individuals legally blind. Hypohidrosis, splenomegaly and arthritis were present in several family members. In contrast to wildtype ALPK1, which is activated specifically by the bacterial metabolite ADP-heptose during bacterial infection, ALPK1[Ser277Phe] was also activated by the human metabolites UDP-mannose and ADP-ribose, even more strongly than the ALPK1[Thr237Met] variant. However, unlike ALPK1[Thr237Met], ALPK1[Ser277Phe] could additionally be activated by GDP-mannose. These observations can explain why these ALPK1 variants are active in cells in the absence of ADP-heptose and hence why patients have episodes of autoinflammation. Examination of the three-dimensional structure of ALPK1 revealed that the sidechains of Ser277 and Tyr254 interact but mutational analysis established that this interaction is not critical for the integrity of the ADP-heptose binding site. Instead, it is the replacement of Ser277 by a large hydrophobic phenylalanine residue or the replacement of Tyr254 by a much smaller cysteine residue that is responsible for altering the specificity of the ADP-heptose-binding pocket. The characterisation of ALPK1 variants that cause ROSAH syndrome suggests ways in which drugs that inhibit these disease- causing variants selectively can be developed.