Mutational analysis in a monozygotic twin pair identified the GNAO1 R209C mutation as a second hit for ETV6-RUNX1⁺ preleukemia. (A) Schematics of a pair of monozygotic twins who were diagnosed with leukemia within 110 days after birth. (B) Schematics of the t(12; 21) ETV6-RUNX1 fusion gene and validation by PCR sequencing. (C) Sanger sequencing of the twins’ samples. GNAO1 c.625C>T point mutations in the twins’ samples at onset of leukemia are indicated: twin A, at the onset of leukemia and at remission; twin B, before the onset of leukemia and at the onset of leukemia. (D) Allele frequency analysis of gene mutations by deep genomic sequencing in the onset specimens from the twins, corresponding to supplemental Table 1. (E) Allele frequency analysis of the ETV6-RUNX1 fusion gene and GNAO1 R209C mutation in twin B before the onset of leukemia.