Nonsynonymous germline sequence variants in the TYK2 gene. (A) Diagram of the TYK2 protein showing nineteen (19) nonsynonymous changes. Four novel germline SNPs (*) were detected in the 94 Discovery set samples. Previously identified SNPs are also shown. Percentages indicate the frequency of the indicated sequence variant in the 94 Discovery set samples. †The TYK2^G363S sequence change is found at a significantly different frequency between AML patients and normal controls. (B) Autophosphorylation of variant TYK2 alleles in response to stimulation by interferon-α (IFN). The human TYK2-deficient cell line U1A was transduced with cDNAs encoding patient-derived variant TYK2 alleles. An artificial allele, V678F, containing an amino acid substitution homologous to the V617F activating mutation in JAK2, was used as a positive control. Most patient-derived alleles were indistinguishable from wild-type. In contrast, TYK2^I684S consistently demonstrated reduced total TYK2 protein levels, whereas the absolute level of phosphorylated TYK2^I684S appeared no different from wild-type (lane 8). Autophosphorylation of the TYK2^P1104V was reduced after IFN stimulation, suggesting a decreased level of kinase activity (lane 12). TYK2^V362F phosphorylation appears slightly increased (lane 5), but this finding was not reproduced in replicate experiments. This experiment was performed 3 times, and a representative blot is shown.