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.