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. 2021 Apr 1;106(4):1188-1192.
doi: 10.3324/haematol.2020.259275.

A homozygous missense variant in UBE2T is associated with a mild Fanconi anemia phenotype

Laura Schultz-Rogers  1 , Francis P Lach  2 , Kimberly A Rickman  2 , Alejandro Ferrer  1 , Abhishek A Mangaonkar  1 , Tanya L Schwab  1 , Christopher T Schmitz  1 , Karl J Clark  1 , Nikita R Dsouza  3 , Michael T Zimmermann  3 , Mark Litzow  1 , Nicole Jacobi  4 , Eric W Klee  1 , Agata Smogorzewska  2 , Mrinal M Patnaik  1
Affiliations

A homozygous missense variant in UBE2T is associated with a mild Fanconi anemia phenotype

Laura Schultz-Rogers et al. Haematologica. .
No abstract available

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Figures

Figure 1.
Figure 1.
The proband carries a likely pathogenic UBE2T variant expressed at low levels conferring defective interstrand crosslink repair. (A) Sequencing of genomic DNA extracted from primary fibroblasts (PM085) of the affected individual indicating a homozygous chr1:202333539G>T variant (hg38, reverse). (B) Sequencing of complementary DNA (cDNA) from the proband’s fibroblasts indicating the presence of a variant NM_014176.3:c.196C>A and no evidence of aberrant splicing. Exon numbering reflects ref seq NM_014176.3 since the primers were designed against this transcript. (C) Immunoblot with anti-UBE2T antibody in whole cell extract from the proband’s primary fibroblasts (PM085), wildtype BJ fibroblasts (from the American Type Culture Collection) and fibroblasts from an UBE2T/FANCT-null Fanconi anemia patient (RA2627). (D) Immunoblot with anti-HA antibody in PM085 (proband) and RA2627 (UBE2T-/-) primary fibroblasts and PM085 EH (immortalized fibroblasts) expressing C-HAFLAG empty vector (EV) or wild-type (WT) UBE2T. HA expression in parental (P) (nontransduced) cells and cells expressing EV or WT UBE2T. (E) Immunoblot with anti-FANCD2 antibody on whole cell extracts of cells treated or not with mitomycin C (MMC). Ub-D2 indicates the monoubiquitinated band. (F) Formation of foci of FANCD2 after MMC treatment in patient-derived PM085 cells (nontransduced parental cells) or cells expressing EV, or WT UBE2T. (G) Cell survival of the proband’s PM085 fibroblasts expressing EV or WT UBE2T after treatment with MMC.
Figure 2.
Figure 2.
P66T UBE2T is a partial loss-of-function variant. (A) Immunoblot with anti-HA antibody of RA2627 (UBE2T-/-) primary fibroblasts expressing empty vector (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 W T UBE2T. (D) Quantification of FANCD2 foci formation af ter MMC treatment in RA2627 (UBE2T-/-) fibroblasts expressing EV, P66T UBE2T, or W T 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 signif icance using one-way analysis of variance with multiple comparisons. ns: not significant, ****P=≤0.0001.
Figure 3.
Figure 3.
UBE2T does not have a major role in the repair of non-interstrand crosslink DNA lesions. (A) Cell survival assay after ultraviolet treatment of complemented pairs of RA2627 fibroblasts compared to BJ wild-type (WT) fibroblasts depleted of XPF used as a positive control. The immunoblot shows decreased XPF levels after siRNA depletion. (B) Cell survival assay of RA2627 fibroblasts after treatment with irradiation. HA239F fibroblasts with RAD50 mutations are sensitive to irradiation and act as a positive control (RAD50mut). (C, D) Cell sensitivity assays comparing RA2627 fibroblasts to RA3331 Fanconi anemia patient-derived fibroblasts with SLX4 mutations (SLX4mut) expressing WT SLX4 or empty vector exposed to camptothecin (C) and the PARP inhibitor, olaparib (D). (E) Cell survival assay after hydroxyurea treatment of RA2627 cells compared to the RA3226 BRCA2 patient cell line (BRCA2mut). Error bars indicate the standard deviation. EV: empty vector; UV: ultraviolet irradiation; IR: irradiation; CPT: camptothecin; PARPi: PARP inhibitor; HU: hydroxyurea.
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References

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