Figure 2.
Germline homozygous LOF mutations in TET2 in 3 immunodeficient patients with lymphoma. (A) Pedigree of 2 unrelated consanguineous families, as well as Sanger sequencing results, of patients and unaffected family members with highlighted affected nucleotide (in bold) in the sequence of TET2. Affected patients, all with homozygous variant, are indicated by filled black symbols, heterozygous family members are indicated by gray symbols, and other relatives are indicated by open symbols. Slashes indicate deceased patients. Arrows above Sanger sequencing traces indicate position of mutated nucleotide (black arrow, heterozygous; red arrow, homozygous variant). The effect on amino acid sequence is shown below the underlined mutated codon. (B) Schematic diagram of TET2 protein structure with highlighted mutated residues H1382R and Q1632* within catalytic double stranded β-helix (DSBH) domain, predicting damaging effect of the mutations on enzyme activity. H1382 residue is positioned in the catalytically important Fe2+-binging HxD motif. (C) TET2 protein expression, as detected by immunoblotting, in fibroblasts of an unrelated control (C), P1, and P2 in Family 1 (upper left panel). Absent TET2 expression in P3 and its decreased expression in heterozygous relatives, as detected by immunoblotting, in PBMCs of Family 2: unrelated control (C), mother (M), father (F), sibling 1 (S1), sibling 2 (S2), and P3 (upper right panel). Quantification of TET2 expression normalized to housekeeping proteins GAPDH and β-actin. Data are mean ± SD from 2 independent experiments. **P < .01, ***P < .001 patient vs respective healthy control and pooled heterozygous relatives vs control, unpaired Student t test. (D) Impaired TET2 hydroxymethylating activity detected by 5hmC immunofluorescence staining in HEK293T cells transfected with empty lentiviral vector, Flag-tagged wt TET2, or mutant TET2H1382R. Blue, DAPI stain; green, Flag; red, 5hmC staining. Result is representative of 3 independent experiments. (E) Increased 5mC/5hmC ratio, as determined by DNA methylation assay of total blood DNA, in patients bearing homozygous H1382R and Q1632* mutations (red bars) compared with homozygous wt controls (black bars). Heterozygous relatives (blue bars) showed significantly increased intermediate levels. Data are mean ± SD from 2 independent experiments and 7 healthy controls. **P < .01, ***P < .001 vs healthy controls, unpaired Student t test. ns, not significant.

Germline homozygous LOF mutations in TET2 in 3 immunodeficient patients with lymphoma. (A) Pedigree of 2 unrelated consanguineous families, as well as Sanger sequencing results, of patients and unaffected family members with highlighted affected nucleotide (in bold) in the sequence of TET2. Affected patients, all with homozygous variant, are indicated by filled black symbols, heterozygous family members are indicated by gray symbols, and other relatives are indicated by open symbols. Slashes indicate deceased patients. Arrows above Sanger sequencing traces indicate position of mutated nucleotide (black arrow, heterozygous; red arrow, homozygous variant). The effect on amino acid sequence is shown below the underlined mutated codon. (B) Schematic diagram of TET2 protein structure with highlighted mutated residues H1382R and Q1632* within catalytic double stranded β-helix (DSBH) domain, predicting damaging effect of the mutations on enzyme activity. H1382 residue is positioned in the catalytically important Fe2+-binging HxD motif. (C) TET2 protein expression, as detected by immunoblotting, in fibroblasts of an unrelated control (C), P1, and P2 in Family 1 (upper left panel). Absent TET2 expression in P3 and its decreased expression in heterozygous relatives, as detected by immunoblotting, in PBMCs of Family 2: unrelated control (C), mother (M), father (F), sibling 1 (S1), sibling 2 (S2), and P3 (upper right panel). Quantification of TET2 expression normalized to housekeeping proteins GAPDH and β-actin. Data are mean ± SD from 2 independent experiments. **P < .01, ***P < .001 patient vs respective healthy control and pooled heterozygous relatives vs control, unpaired Student t test. (D) Impaired TET2 hydroxymethylating activity detected by 5hmC immunofluorescence staining in HEK293T cells transfected with empty lentiviral vector, Flag-tagged wt TET2, or mutant TET2H1382R. Blue, DAPI stain; green, Flag; red, 5hmC staining. Result is representative of 3 independent experiments. (E) Increased 5mC/5hmC ratio, as determined by DNA methylation assay of total blood DNA, in patients bearing homozygous H1382R and Q1632* mutations (red bars) compared with homozygous wt controls (black bars). Heterozygous relatives (blue bars) showed significantly increased intermediate levels. Data are mean ± SD from 2 independent experiments and 7 healthy controls. **P < .01, ***P < .001 vs healthy controls, unpaired Student t test. ns, not significant.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal