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Letters to the Editor
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Figure 2. P66T UBE2T is a partial loss-of-function variant. (A) Immunoblot with anti-HA antibody of RA2627 (UBE2T-/-) primary fibroblasts expressing empty vec- tor (EV) or C-HA-FLAG P66T UBE2T or wild-type (WT) UBE2T. (B) Cell survival of RA2627 (UBE2T-/-) fibroblasts expressing EV, P66T UBE2T, or WT UBE2T after treatment with mitomycin C (MMC). (C) FANCD2 ubiquitination with and without MMC treatment in RA2627 (UBE2T-/-) fibroblasts expressing EV, P66T UBE2T, or WT UBE2T. (D) Quantification of FANCD2 foci formation after MMC treatment in RA2627 (UBE2T-/-) fibroblasts expressing EV, P66T UBE2T, or WT UBE2T. Approximately 300 HA-expressing cells were analyzed for the presence of FANCD2 foci in three separate coverslips. The mean percent nuclei with FANCD2 foci was plotted and tested for significance using one-way analysis of variance with multiple comparisons. ns: not significant, ****P=≤0.0001.
interstrand crosslinking agent mitomycin C. Normal FANCD2 monoubiquitination was observed in the WT control cell line (BJ fibroblasts), was absent in UBE2T-/- (RA2627) and FANCA-/- (RA3087) fibroblasts and was reduced in the proband’s fibroblasts (Figure 1E). Expression of WT UBE2T in the patient’s fibroblasts fully rescued FANCD2 monoubiquitination (Figure 1D and E), recruitment of FANCD2 to chromatin after mitomycin C treatment, and sensitivity of the proband’s fibroblasts to mitomycin C (Figure 1F-H). These results indicate that the proband belongs to FA-T complementation group and suggest that the patient’s missense variant is hypo- morphic, resulting in reduced function.
To further demonstrate that the missense variant reduces UBE2T function and is indeed likely pathogenic, UBE2T-/- cells were transduced with either WT or P66T HA-tagged UBE2T (Figure 2A). The P66T variant expressed at a lower level compared to WT UBE2T, con- sistent with decreased stability of UBE2T carrying that variant. Expression of P66T UBE2T also only partially rescued cell survival, FANCD2 ubiquitination, and FANCD2 foci formation upon treatment with mitomycin C compared to WT UBE2T expression (Figure 3B-D). This provides further evidence that the missense variant is a likely pathogenic hypomorph.
The cellular and patients’ phenotypes described for the
FA-T complementation group are thus far consistent with defective FA pathway activation and a defect in interstrand crosslink repair. However, it was previously reported that UBE2T-deficient DT40 cells were sensitive to ultraviolet irradiation and the replication stress-inducing agent, hydroxyurea.11 To determine whether UBE2T is important for resistance to other types of DNA damage, RA2627 cells were tested for sensitivity to a number of other genotoxic agents. RA2627 cells were not found to be hypersensitive to ultraviolet irradiation, ionizing radiation, camptothecin, hydroxyurea, or the PARP inhibitor olaparib (Figure 3A-E). These data suggest that UBE2T does not have a major role in responding to DNA lesions or replication stress pro- duced by these agents and its primary function is in inter- strand crosslink repair and that the patients’ phenotypes reflect defects in the repair of interstrand crosslink lesions.
In conclusion, we report a novel presentation of FA-T complementation group resulting from a likely pathogen- ic missense variant (c.196C>A) in UBE2T. The patient presented with atypical, mild FA, characterized by per- sistent macrocytosis and neutropenia with intermittent thrombocytopenia but no severe bone marrow failure (without evidence of somatic reversion in blood) or con- genital abnormalities common to FA. Clinical chromoso- mal breakage assays were consistent with a diagnosis of FA and subsequent functional analysis of patient-derived
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haematologica | 2021; 106(4)