Figure 1.
Functional characterization of pre-TCRα variants truncating the intracellular domain. (A) Pedigrees of the five kindreds carrying homozygous premature stop or frameshift variants in the intracellular domain of the pre-TCRα chain encoded by PTCRA (NM_138296.3). P2 and P3 were diagnosed with RPGRIP1 deficiency (NM_020366.4: c.1107del; p.Glu370Asnfs*5). P4 and P5 had a suspected diagnosis of CUL7 deficiency (NM_014780.5: c.3807-18G>A; splice AI Δ score for an acceptor gain = 0.53). P6 was diagnosed with STK4 deficiency (NM_006282.5: c.871C>T; p.Arg291*). The PTCRA variants appear in red, whereas the variants of other genes appear in blue. M: mutant allele, E?: unknown genotype (B) Schematic representation of the PTCRA isoform A protein (2), indicating the location of the variants studied. The variants from the patients appear in red. The artificial variants created in this study appear in purple. SP, signal peptide; TM, transmembrane domain. (C) HEK293T cells were transfected with an empty vector (EV), or with plasmids encoding the WT or indicated PTCRA isoform A variants. Total protein extracts were analyzed by immunoblotting with an antibody against pre-TCRα or vinculin (loading control). (D and E) TCRα-deficient Jurkat cells were transduced with EV or with plasmids encoding WT or mutant pre-TCRα isoform A. CD3ε surface expression was assessed by flow cytometry. (D) Bar graphs showing mean fluorescence intensity (MFI) for CD3ε. (E) Representative flow cytometry histograms. In each histogram, the trace of cells transduced with the EV is shown in grey, and the trace of cells transduced with the indicated variant is shown in red. All results are representative of three independent experiments. The methods used in C–E have been described in detail elsewhere (2). Source data are available for this figure: SourceData F1.

Functional characterization of pre-TCRα variants truncating the intracellular domain. (A) Pedigrees of the five kindreds carrying homozygous premature stop or frameshift variants in the intracellular domain of the pre-TCRα chain encoded by PTCRA (NM_138296.3). P2 and P3 were diagnosed with RPGRIP1 deficiency (NM_020366.4: c.1107del; p.Glu370Asnfs*5). P4 and P5 had a suspected diagnosis of CUL7 deficiency (NM_014780.5: c.3807-18G>A; splice AI Δ score for an acceptor gain = 0.53). P6 was diagnosed with STK4 deficiency (NM_006282.5: c.871C>T; p.Arg291*). The PTCRA variants appear in red, whereas the variants of other genes appear in blue. M: mutant allele, E?: unknown genotype (B) Schematic representation of the PTCRA isoform A protein (2), indicating the location of the variants studied. The variants from the patients appear in red. The artificial variants created in this study appear in purple. SP, signal peptide; TM, transmembrane domain. (C) HEK293T cells were transfected with an empty vector (EV), or with plasmids encoding the WT or indicated PTCRA isoform A variants. Total protein extracts were analyzed by immunoblotting with an antibody against pre-TCRα or vinculin (loading control). (D and E) TCRα-deficient Jurkat cells were transduced with EV or with plasmids encoding WT or mutant pre-TCRα isoform A. CD3ε surface expression was assessed by flow cytometry. (D) Bar graphs showing mean fluorescence intensity (MFI) for CD3ε. (E) Representative flow cytometry histograms. In each histogram, the trace of cells transduced with the EV is shown in grey, and the trace of cells transduced with the indicated variant is shown in red. All results are representative of three independent experiments. The methods used in C–E have been described in detail elsewhere (2). Source data are available for this figure: SourceData F1.

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