Novel mutations identified by WES or targeted NGS lead to TYK2 deficiency
The mutations were identified by whole exome sequencing (WES) for P1, P3 and P5 or targeted next generation sequencing (NGS, PID gene panel) for P2 and P4 (Fig. 1A-1C). P1 carried novel compound heterozygous mutations, c.3041T>C, p.L1014P (MAF: EXAC 0.000008; CADD score 32, pathogenic) and c.1253C>A, p.S418X (pathogenic) in TYK2 (Fig. 1D). P2 carried a novel homozygous mutation, c.2395G>A, p.G799R (MAF: EXAC 0.000008; CADD score 28.5, pathogenic) in TYK2 (Fig. 1D). P3 carried novel compound heterozygous mutations, c.1621G>A, p.G541R (MAF: EXAC 0.000058; CADD score 23, pathogenic) and c.704G>A, p.R235Q (MAF: EXAC 0.000114653; CADD score 22.4, pathogenic) in TYK2 (Fig. 1D). P4 carried compound heterozygous mutation, c.2269C>G, p.L757V (MAF: no recorded; CADD score 33, pathogenic) and c.149delC p. S50CfsX2 (pathogenic) in TYK2 (Fig. 1D). P5 carried a novel homozygous mutation, c.1507C>T, p.R503X (pathogenic) in TYK2 (Fig. 1D). These mutations were all further confirmed by Sanger sequencing (Fig. 1D). According to inhouse prediction algorithm developed by the sequencing companies, these mutations were all predicted to be pathogenic to the expression or function of TYK2 protein (Fig. 1B and 1C). To confirm this, we then determined the protein level of TYK2 in patients’ PBMCs and we found almost abolished expression of TYK2 (Fig. 1E-1H) in all patients except for P4, whose blood sample was not available. The anti-TYK2 antibody recognizes 200-400 amino acids of TYK2. We also determined the mRNA level of TYK2 in P1, P2 and P3, we found that their mRNA levels were decreased (Fig. 1I-1K).
To further understand and confirm the pathogenicity of these mutations, we performed Cycloheximide Chase Assay to test the protein stability. We found that these mutations indeed impaired the protein stability (SupFig. 2). Please note that mutation in P2 has been tested to impaired the protein stability previously[8]. Taken together, these five patients from five unrelated families all had TYK2 deficiency.